Evaluation of the biological responses of osteoblast-like UMR-106 cells to the engineered porous...

Evaluation of the biological responses of osteoblast-likeUMR-106 cells to the engineered porous PHBV matrix

Hui Liu,1 Dharmaraj Raghavan,1 John Stubbs III21Polymer Group, Department of Chemistry, Howard University, Washington District of Columbia 200592Department of Microbiology, College of Medicine, Howard University, Washington District of Columbia 20059

Received 26 July 2006; revised 22 August 2006; accepted 5 September 2006Published online 22 December 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.31101

Abstract: Poly (3-hydroxybutyrate-co-3-hydroxyvalerate)(PHBV) has been investigated for biomedical applicationsdue to its many biologically favorable properties. How-ever, to explore its application in bone tissue engineering,the poorly bioactive surface property of PHBV must beimproved. To engineer PHBV to achieve a biologicallyactive surface, in this study each porous PHBV matrix wasprepared by solute leaching of salt/PHBV cast film andwas treated with ozone followed by dip coating with typeI collagen. The biological responses of osteoblast-likeUMR-106 cells after being grown on the engineered PHBVmatrix were evaluated. Confocal microscopy and the MTTassay were used to map and quantify the viable cell prolif-eration on the PHBV matrix, respectively. The cells werecultivated in osteogenic media containing b-glycerophos-phate and later stained with alizarin red to visualize min-

eralization of the matrix. RNA was extracted from theUMR-106 cells, and reverse transcriptase-polymerase chainreaction (RT-PCR) was applied to detect expression ofglyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ahouse keeping gene) and bone sialoprotein (BSP) (markerof the osteoblastic phenotype). The results showed that theUMR-106 cells after cultivation on the engineered PHBVmatrix retained the osteoblastic phenotype characteristics,indicating that the porous PHBV matrix after ozone treat-ment and collagen dip coatings are a promising scaffoldfor bone tissue engineering applications. � 2006 WileyPeriodicals, Inc. J Biomed Mater Res 81A: 669–677, 2007

Key words: bone tissue engineering; biocompatibility; cellviability; collagen; osteogenesis

INTRODUCTION

Although a number of polymers have been designedand investigated for biomedical and pharmaceuticalapplications 1–4 poly (3-hydroxybutyrate-co-3-hydroxy-valerate) (PHBV) is still under extensive investigationbecause of its biologically favorable properties. PHBVis a natural biodegradable polymer that recently hasreceived attention in the field of bone tissue engineer-ing.5–9 The interest in using PHBV for bone tissue engi-neering applications is in part due to the reason thatPHBV can be readily tuned to achieve desired mechan-ical properties and degradation rate.10–12 In addition,

PHBV possesses biologically favorable characteristicsfor in vivo application, such as low immunogenicity,nontoxicity to living tissues, and predictable biodegra-dation kinetics.13–19 Moreover, PHBV has also beenshown to support fibroblast cell growth similarly tothat noticed in collagen sponges.9,20

However, there are certain problems that have lim-ited the use of PHBV in bone tissue engineering. Onemajor problem is that PHBV lacks surface bioactivity topromote attachment of tissues to the polymer ma-trix.9,12,21–27 For a given environment, cell attachmentto a polymer is strongly dependent on the surface char-acteristics (i.e. topography and chemistry) of the poly-mer.12,28,29 Because of the absence of natural cell recog-nition, sites on PHBV, surface treatment techniquesthat can functionalize the polymer surface, must besought to promote favorable cellular and physiologicalresponses.30

As PHBV is poorly wettable, improving the wettabil-ity of the PHBV matrix has been attempted usingplasma or g radiation or ozone-induced oxida-tion.9,12,31,32 Such approaches have endowed the solidpolymer surface with rich chemical functionality, whilekeeping the polymer surface flat on the micrometer

Correspondence to: D. Raghavan; e-mail: [email protected] grant sponsor: Keck Foundation, NSF; contract

grant number: DMR-0213695Contract grant sponsor: U.S. Army Medical Research Ma-

terial Command; contract grant number: DAMD17-01-1-0268Contract grant sponsor: NIH; contract grant number:

S06GM008016-35

' 2006 Wiley Periodicals, Inc.

scale.33 To make the chemically modified PHBV morebioactive, one approach is to coat biological moleculesonto the polymer surface.9,12 One of the benefits ofcoating biological molecules on the polymer surface isto promote cell attachment.34,35 For example, it wasreported that solid surfaces coated with peptidesresulted in enhanced cell attachment, 36,37 as cell attach-ment is attributed to the interactions between theextracellular matrix proteins adsorbed on the polymersurface and the adhesion receptors on the cell mem-brane.38 Cell attachment can also be promoted by cova-lently bonding peptides to the polymer surface usingchemical immobilization methods. In addition,improved cell attachment can be acquired by increas-ing the surface area of the polymer matrix so as toimprove cell anchoring and transfer of nutrients andcellular media across the matrix material.39,40

In this study, we first prepared a macroporousthree-dimensional (3D) PHBV matrix by solute leach-ing of salt/PHBV cast film to increase in surface area.We then employed a comprehensive approach to engi-neer the porous PHBV matrix to make it more surfacebioactive. We treated the PHBV matrix with ozone toincrease its surface hydrophilicity and dip coated theozone-treated matrix with collagen to enhance its sur-face cell-binding ability because collagen has cell-bind-ing domains containing the RGD (Arg-Gly-Asp) andDGEA (Asp-Gly-Glu-Ala) sequences that are essentialfor cell adhesion.34 To determine whether such treat-ments could result in a surface bioactive PHBV matrix,we chose osteoblastic-like UMR-106 cells as a modeland examined their biological response on the engi-neered porous PHBV matrix. Tissue culture poly-styrene (TCPS) was used as a control substrate forcomparison.

MATERIALS AND METHODS

Materials

Poly (3-hydroxybutrate-co-3-hydroxyvalerate) (PHBV,containing 8 wt % hydroxyvalerate), type I collagen derivedfrom calf skin, 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyl tetra-zolium bromide (MTT) were purchased from Sigma-Aldrich(St. Louis, MO). Glyceraldehyde 3-phosphate dehydrogen-ase (GAPDH) primer pairs (U: 5-ACT TTG TCA AGC TCATTT CC-3 D: 5-TGC AGC GAA CTT TAT TGA TG-3),41 andbone sialoprotein (BSP) primer pairs (U: 5-GAA ACG GTTTCC AGT CCA G-3 D: 5-TGA AAC CCG TTC AGA AGG-3)42 were purchased from Sigma-Genosys (St. Louis, MO).Access RT-PCR system (Nuclease-Free water, AMV/TflReaction Buffer, dNTP mix, upstream primer, downstreamprimer, MgSO4, AMV Reverse Transcriptase, and Tfl DNAPolymerase) and SV Total RNA Isolation System (RNALysis Buffer(RLA), RNA Dilution Buffer (RDA), RNA WashSolution (RWA), and Dnase Stop Solution (DSA) wereobtained from Promega Corporation (Madison, WI). TBE

polyacryamide gels (6%) were obtained from Invitrogen(Carlsbad, CA). Safefix II was obtained from Fisher Scientific(Fair Lawn, NJ) and DNA LADDER I was obtained fromGenechoice (Frederick, MD). Alizarin Red-S(ARS) was pur-chased from Acros Organics (Fisher Life Sciences). Rat os-teosarcoma UMR-106 (ATCC, CRL-1661) cell line was pur-chased from American Type Culture Collection. 6-well cellculture plates (Corning No. 3516) and 96-well cell cultureplates were purchased from Costar (Costar No. 3596).

Preparation of macroporous PHBV films

A combination of the solvent casting and solute leachingtechniques was used to prepare macroporous PHBV films.Sieved sodium chloride (NaCl) was mixed with 1.0 g ofPHBV powder manually, and then the mixture was dis-solved in 10 mL of chloroform at 608C. The salt/PHBV solu-tion was cast in a precleaned glass petri-dish and the solventwas allowed to gradually evaporate over 24 h at room tem-perature. Vacuum was applied to remove solvent remnantsin the film for 24 h at room temperature. Then, the PHBV/NaCl composite film was released from the Petri-dish andmechanically agitated at room temperature for several days,during which the water was replaced every 2 h for the first8 h and then two to three times a day to remove free ions.The washing was continued until the solution was free ofchloride ions by checking the aliquot filtrate solution with0.1N AgNO3 solution. The salt-leached PHBV film was vac-uum dried for 24 h and stored in a dessicator prior to use.Micrometer was used to measure the thickness of the pre-pared porous films. Some dried film were used for opticaland AFM characterization while the remaining films wereused for cell proliferation measurement.

Preparation of ozone-treated and collagen-coatedmacroporous PHBV films

Macroporous PHBV films of thickness 340 mm were cutinto circular pieces with a diameter of 10 and 32 mm,respectively. The disc-shaped PHBV films were placed in aPyrex fritted glass chamber and subject to ozone treatmentto increase the surface hydrophilicity of the films. Ozonewas generated at room temperature by passing oxygen at arate of 2.2 g/h through an ozone generator (Ozonology,Model no. L-25, Evanston, IL). The ozone along with oxy-gen was allowed to sweep the films for 20 min at roomtemperature. After discontinuing the ozonolysis experiment,fresh oxygen was allowed to purge for a brief duration(10 min) so that the PHBV film could be flushed of unreactedozone. Then the ozone-treated PHBV films were removed andimmediately dipped into a type I collagen solution (4 mg/mLin 0.3% acetic acid) for 24 h at 2–48C. The collagen-coated filmswere then air-dried for 24 h at 2–48C.

Cell culture of osteoblast-like UMR-106 cells

Osteoblast-like UMR-106 cells were cultured in Dulbec-co’s Modified Essential Medium (DMEM) supplementedwith 10% (v/v) heat inactivated fetal bovine serum (FBS),

670 LIU, RAGHAVAN, AND STUBBS

Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a

2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mLstreptomycin (Quality Biological) at 378C in 5% CO2. Forsubculture, the cells were harvested and washed withHank’s balanced salt solution (Sigma-Aldrich) and trypsi-nized with Trypsin-EDTA (0.05 % trypsin, 0.1% EDTA)(Quality Biological) for 15 min at 378C to obtain a cell sus-pension. Trypsin activity was inhibited upon the additionof FBS at a final concentration of 10%. The cells were thencentrifuged and washed with serum-free DMEM threetimes to remove residual FBS. The relative centrifugal forceused was *150g. The concentration of the resulting cellsuspension was determined with a hemocytometer.

Cell proliferation on the collagen-coatedPHBV films

For the cell activity study, 3.0 � 104 osteoblast-likeUMR-106 cells were seeded onto a collagen-coated macro-porous PHBV film, which was previously placed in a 96-well plate. After seven days incubation at 378C in 5% CO2,the cells were washed twice with serum free DMEM. TheMTT assay was used to measure the metabolic activity ofthe cells. Briefly, 100 mL serum free medium and 100 mL of5 mg/mL MTT solution were added to the film, and thenincubated at 378C for 4 h to form MTT formazan. After-ward, 200 mL DMSO was added to the film for 3 h to dis-solve the MTT formazan crystals. MTT formazan wasrecovered by dissolving in 100 mL of DMSO mixture andthe absorbance was measured by a Dynatech laboratories’Microplate Reader 5000/7000 at the wavelength of 570 nm.

Confocal microscopy characterization

The viable cells invading into the PHBV film were fluo-rescently labeled by adding 5 mg/mL 20,70-bis-(2-carbox-yethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester(BCECF-AM) in complete media to the film. The mixturewas incubated for 1 h followed by rinsing in PBS solution.The film was retrieved, air dried, and then examined usingan upright laser scanning confocal microscope (Olympus).The fluorescence adsorbed cells were excited at 488 nmusing Arþ laser light, and light emitted (505–550 nm) fromthe film was detected with a photomultiplier tube. Imageswere acquired by focusing the laser beam at focal planesbeneath the surface of the film.

RNA analysis

RNA was extracted from the cells grown on the PHBVfilm and compared with the RNA extracted from cellsgrown on TCPS for analysis of the osteoblastic phenotypecharacter. Briefly, 1.4 � 106 cells were incubated into eachwell of a 6-well plate covered by a collagen-coated PHBVfilm. Total cellular RNA was isolated with the SV TotalRNA Isolation System using the vacuum purification proto-col provided by the manufacturer. The 10-day incubatedTCPS and PHBV samples were homogeneously mixed inRLA. The solution was transferred to a clean tube, mixedwith RDA, and then centrifuged. The supernatant solution

was withdrawn and mixed with 95% ethanol, and was cen-trifuged again. The relative centrifugal force was *19,000g.The clear supernatant solution was placed in a spin columnassembly, and vacuum was applied to remove the lysate.Vacuum was also applied upon adding RWA to the spincolumn assembly. The spin column assembly was treatedwith DSA for 15 min prior to the application of vacuum.Finally, the spin column assembly was washed twice withRWA, and transferred to a tube for centrifugation to recoverthe RNA.

RT-PCR was applied to amplify the recovered RNAfrom tissues. One microliter of the isolated RNA mixturewas added to a mixture solution containing 49 mL of RT-PCR master mixture (27.4 mL Nuclease-Free water, 10 mLAMV/Tfl Reaction Buffer, 1 mL dNTP mixture, 3.3 mL of40 mM specific upstream primer, 3.3 mL of 40 mM down-stream primer, 2 mL of 25 mM MgSO4, 1 mL of 5 units/mLAMV Reverse Transcriptase, and 1 mL of 5 units/mL TflDNA Polymerase). The primers used in this study wereGAPDH (a house keeping gene) and BSP (a marker of theosteoblastic phenotype). Following the manufacturer’sprotocol, the total RNA was amplified with the AccessRT-PCR system. The RT-PCR reaction was carried out at458C for 45 min to synthesize the first strand cDNA byreverse transcription, subsequently exposed to 948C for2 min to inactivate the AMV/RT and denature the RNA/cDNA complex. The PCR process was repeated for 40cycles (948C for 30 s, 608C for 1 min, and 688C for 2 min)to amplify the specific cDNA. The amplified cDNA prod-uct was subject to 688C for 7 min and then stored at 48C.Then 10 mL of cDNA solution was mixed with 5 mL ofgel running reagent. The combined solution was heatedto 758C for 3 min in a water bath. About 15 mL of eachsample and 7 mL of the prepared DNA Ladder I (sizemaker) were settled into the GEL’s wells. Gel electrophore-sis was performed in 1 � TBE electrophoresis buffer (TrisBorate EDTA) at 200 V and 3.0 Amper for 28 min. Ethi-dium bromide staining was utilized to visualize the ampli-fied DNA.

Mineralization study

Cells/well (3.0 � 104) were seeded in the 96-well plates.Cells were grown in DMEM supplemented with 10% (v/v)heat inactivated FBS, 2 mM L-glutamine, 100 U/mL peni-cillin, and 100 mg/mL (w/v) streptomycin for seven days.Then calcium mineralization was induced by addition ofDMEM containing 50 mg/mL L-ascorbic acid, 10% FBS,and 10 mM b GP for an additional seven days. The cellswere cultured at 378C with 5% CO2. The growth mediumwas changed every three days for 14 days. After 14 days,the films in the 96-well plates were withdrawn, washedwith PBS, and fixed by Safefix II at room temperature forabout 15 min. Deionized (DI) water was used to wash thefilms twice. About 150 ml of 40 mM fresh Alizarin Red S(pH adjusted to 4.35 by 0.5% ammonium hydroxide) wasadded to each well. The samples were incubated for20 min at room temperature. The unbonded dye was aspi-rated out. The wells were washed between six and eighttimes with 200 mL DI water while shaking. The stainedfilms were visualized with a camera HP Photosmart 735.

THE BIOLOGICAL RESPONSES OF UMR-106 CELLS TO ENGINEERED POROUS PHBV 671


RESULTS AND DISCUSSION

Morphology of the prepared PHBV films

Figure 1 shows the optical micrograph of thePHBV film prepared by salt leaching of PHBV/NaClover the duration of five to eight days. It can benoticed that the salt particulates after leaching pro-duced pores on the film surface. The characteristicdimension of the pores ranged from 45 mm toaround 150 mm, which was consistent with the saltparticle dimension used in the study.

A three-dimensional AFM topographic image ofthe porous PHBV film is shown in Figure 2. As illus-trated in the figure, a valley corresponding to the‘‘pits’’ exists within a 50 mm � 50 mm area of thefilm. The pit formation observed in this study wasconsistent with the pores resulting from the salt par-ticulate leaching method.9,43

In this study, the procedure used for preparationof porous films of a single salt composition wasalso applied to prepare porous films of varyingdimensions. Membranes of 20 wt %, 40 wt %, and60 wt % salt loaded PHBV film, and particle size ofsalt ranging from <45 mm to around 150 mm werecasted. We have previously shown that the inter-connectivity of the pores is related to the particu-late composition and particle size range.21,44 Whenthe leachable component of the sample exceeds 40wt % of the overall weight of the composite, a sig-nificant amount of the leachable component in theparticulate composite is leached leaving behindPHBV film exhibiting macroporous structures withinterconnected open pores.

Cell proliferation ascertained by the MTTassay and confocal microscopy

Cell viability was determined with the MTT assayand the BCECF-AMfluorescence method. 3� 104 cells/cm2 were seeded on the porous PHBV membranesof various dimensions and incubated in serum freemedium for 20 h. Afterward, the cells were trans-ferred to a well containing serum included mediaand allowed to grow for seven days. At the end ofthe seventh day, PHBV films were reacted withMTT. Viable osteoblastic cells converted MTT to aMTT formazan adduct, which was measured at thewavelength of 570 nm.45–47

Figure 3 represents the recorded absorbance dataof the MTT formazan for cells grown on the PHBVfilm of varying (a) film thickness, (b) porosity, and(c) pore volume fraction. The data were an averageof at least three measurements. We reconfirmed ourearlier result21 that the absorbance value of the MTTformazan obtained from the cells on the PHBVmatrix was lower than that on the TCPS. This sug-gests that TCPS shows improved cell viability andgrowth rate relative to PHBV films.48,49 A compari-son of the MTT absorbance data for cells grown onporous PHBV film suggests that there is an optimalthickness (340 mm), porosity (*150 mm), and porevolume fraction (60 wt %) of PHBV film for whichthe osteoblastic cells showed improved cell activityamong the studied systems. For example, weobserved higher cell activity on 60 wt % porousPHBV film compared to 20 wt % porous PHBV filmand this can be attributed to the existence of multi-ple continuous pore pathways in the film. As men-tioned previously, the presence of continuous path-ways facilitates the transport of nutrients across

Figure 1. Phase microscopy of the PHBV scaffolds aftersalt leaching. The image was obtained with a NikonEclipse TE2000-S inverted microscope equipped with a�20 Apodized Dark Low phase contrast lens and a SpotRT Monochrome digital video camera (Diagnostic Instru-ments Sterling Heights, MI).

Figure 2. Three dimensional topographic image ofthe surface of the porous PHBV film after salt leachingfrom salt/PHBV film scan dimension of 120 mm � 120 mm.[Color figure can be viewed in the online issue, which isavailable at www.interscience.wiley.com.]



the film, hence improving cell activity. In addition,3D porous PHBV has higher surface area, whichallowed more cells to anchor on the porous scaf-fold.

To confirm that viable cells have indeed prolifer-ated on the porous membrane, seven day incubatedPHBV film was stained by BCECF-AM and imagedwith confocal microscopy. The BCECF-AM fluores-cence method determined cell viability based on theintracellular esterase activity. Cellular esterases inviable cells convert the non fluorescent BCECF-AMinto the fluorescent 20,70-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) compound. The fluo-rescence images were acquired by focusing the laserbeam at different focal planes along the film thick-ness. Figure 4 shows confocal images of the stainedPHBV film. The green color is a qualitative represen-

tation of the presence of viable cells in the regionsince the local fluorophore environment (presence ofviable cells) influences the fluorescence intensity.7

The brighter area indicates a higher intensity of fluo-rescence. A high intensity of green color representscells with high enzymatic activity and/or many via-ble cells with moderate enzymatic activity. To gener-ate a contour map in terms of the extent of prolifera-tion of osteoblastsic cells on porous PHBV film alongfilm thickness, a quantitative correlation between flu-orescence intensity and the cell activity must beestablished. Future work will delve into this subject.The initial results indicate that osteoblastic cells haveproliferated in the three dimensional matrix andwere viable. The imaging results obtained from theconfocal microscopy were consistent with the MTTassay data.

Figure 3. Cell proliferation as measured by absorbance on porous PHBV film of varying (a) film thickness, (b) porosity,and (c) pore volume fraction. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]



mRNA expression and mineralization

Cells were cultivated in osteogenic media contain-ing beta-glycerophosphate and allowed to grow onthe 3D porous PHBV matrix and TCPS for minerali-zation and mRNA analysis.

Mineralization of the cells grown on the TCPS andPHBV for 14 days was tested by using the Alizarinred staining method as mentioned in the methodssection. Briefly, cultures were grown initially forseven days in serum containing medium, then trans-ferred to a serum containing medium supplementedwith beta glycerol phosphate for another seven days.Figure 5 is a photo micrograph of an alizarin redstained PHBV film and positive control TCPS. In thepresence of osteogenic supplements, densely stainedUMR106 nodules appeared from and within the ma-trix of both TCPS and PHBV film by day 14. Further-more, ARS staining also allowed evaluation of min-

eral distribution within the PHBV film and TCPS. Atpresent the exact mechanism by which beta-glycerolphosphate induces mineralization is not clear; how-ever, it is known from a number of studies that or-ganic phosphates are enzymatically cleaved by alka-line phosphatase to release phosphate ion in solution,which automatically leads to calcium phosphate dep-osition.50–53 Alizarin red-S staining complexes withcalcium phosphate yield reddish brown deposits. Inour system the intensity of the coloration may be pro-portional to the amount of calcium phosphate depos-ited or the level of alkaline phosphatase activity inthe viable osteoblastic cells. Comparison of the micro-graphs suggests that both matrices support calciumphosphate deposition as indicated by the stain. Quali-tatively, the observed coloration of PHBV and TCPSsuggests that both polymeric substrates are responsi-ble for the observed phenotypic expressions noticedduring the assay. Quantitative comparison of the in-

Figure 4. Confocal microgragh of UMR-106 cells grown on collagen dip coated PHBV matrices. Micrograph taken at thesurface (0 mm) (a) and beneath the surface at 82 mm (b), 103 mm (c), 134 mm (d), and 200 mm (e). Strong fluorescence inten-sity was observed at area (A) in Figure 4(a) and area (B) in Figure 4(d) indicating highly viable cells and/or high enzymeactivity. The lack of fluorescence intensity at area (B) in Figure 4(a) and area (A) in Figure 4(d) suggests the presence offar fewer viable cells and/or low enzyme activity as detected by fluorescence BCECF. [Color figure can be viewed in theonline issue, which is available at www.interscience.wiley.com.]



tensity data of the stained PHBV and TCPS will beexplored in our future study.

RT-PCR was performed on the total RNAextracted from the cells followed by electrophoreticanalysis of the PCR product. The expression for twodifferent markers were examined namely GAPDH (ahouse keeping gene) and BSP (a marker of the osteo-blastic phenotype). In our study, TCPS served as thepositive control and deionized water free of mRNAserved as the negative control. Figure 6 shows RT-

PCR data of standard, and mRNA data for osteo-blast cells grown on PHBV and TCPS specimen.Multiplicate samples were used for each specimen tovalidate the data. The primers sequences were con-firmed by the BLAST analysis. For cells cultivatedon 3D PHBV matrix or TCPS, the RT-PCR analysisusing these primers gave an expected fragment of267 bp and 564 bp that correspond to the GAPDHand the bonafide BSP transcripts, respectively. Thissuggests that the matrices support the growth ofosteoblastic-like UMR-106 cells. The amplification ofthe bands was found to be weaker in the PHBV ma-trix than TCPS. In the absence of the quantitativedata of mRNA levels, any differences in the amplifi-cation of transcripts is difficult to interpret. Furtherstudies are underway to collect time-based quantifi-able mRNA data from the cells grown on TCPS andPHBV matrices. In summary, the RT-PCR datashows that the UMR-106 cells cultivated on the po-rous PHBV matrices did not lose their osteoblasticphenotype character and exhibited a favorable prolif-eration rate.

CONCLUSIONS

A conventional salt leaching procedure was usedto prepare three dimensional porous PHBV films.The optical and AFM images confirmed the poreswere produced in the PHBV film by solute leaching.The confocal microscopy and MTT results showedthat the porous PHBV film provided a favorable ma-trix for osteoblast-like cell proliferation. In particular,the MTT assay indicated that the size and volume

Figure 5. Digital photograph of Alizarin Red S stain.UMR-106 cultures were stained with Alizarin Red S cal-cium stain at the end of the experimental period (day 14).(a) Mineral deposition on TCPS. Column M: cells withoutARS staining; column 1, column 2, and column 3: cells af-ter ARS staining. (b) Mineral deposition on PHBV matrixColumn M: cells without ARS staining; column 1, column2, and column 3: cells after ARS staining. [Color figure canbe viewed in the online issue, which is available atwww.interscience.wiley.com.]

Figure 6. RT-PCR analysis (a) Amplification of mRNA extracted from PHBV matrix and TCPS with primer BSP (amarker of osteoblastic phenotype). Lane M: molecular size marker; lane 1: negative control; lane 2: mRNA extracted fromUMR-106 cells grown on TCPS; lane 3: mRNA extracted from UMR-106 cells grown on PHBV matrix. (b) Amplification ofmRNA extracted from PHBV matrix and TCPS with primer glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ahouse keeping gene). Lane M: molecular size marker; lane 1: mRNA extracted from UMR-106 cells grown on TCPS; lane2: mRNA extracted from UMR-106 cells grown on PHBV matrix; lane 3: negative control.



fraction of the pores influenced cell proliferation.According to the comparison of the MTT absorbancedata for cells grown on the porous PHBV films ofvarious dimensions, the PHBV matrix having anoptimal thickness (340 mm), porosity (150 mm), andpore volume fraction (60 wt %) can support for opti-mal osteoblastic cell growth in our system. The con-focal imaging results showed the osteoblast-like cellskept viability and proliferated in the 3D matrix. Min-eralization was observed on the PHBV film andTCPS. The RT-PCR results confirmed the expressionof GAPDH and BSP in the cells grown on the PHBVand TCPS, indicating that the engineered PHBV ma-trix is a biologically favorable scaffold for the cul-tured osteoblast-like cells to maintain their pheno-type characteristics.

Mr. Biamiam Kifle is acknowledged for providing com-ments on the cell culture of osteoblast-like UMR-106 cellsand Dr. K. C. Prabha in assisting with confocal micros-copy.

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Evaluation of the biological responses of osteoblast-like UMR-106 cells to the engineered porous...

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