Biological response of osteoblast-like UMR-106 cells to the modified PHBV matrix—Effects of...

Biological response of osteoblast-like UMR-106 cells to themodified PHBV matrix—Effects of porosity and collagendip coating

Hui Liu,1 Dharmaraj Raghavan,1 Samuel Melaku,1* John Stubbs III21Polymer Group, Department of Chemistry, Howard University, Washington DC 200592Department of Microbiology, College of Medicine, Howard University, Washington DC 20059

Received 24 January 2008; revised 15 September 2008; accepted 31 October 2008Published online 16 March 2009 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32427

Abstract: In this study, we related porosity and collagencoating of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)(PHBV) scaffold to the degree of cell proliferation on theengineered PHBV scaffold. Based on the [3-(4,5-dimethylth-iazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium, inner salt] (MTS) assay, we established thatUMR-106 cell proliferation is maximum in collagen-coated porous PHBV film followed by porous PHBV filmand least in nonporous PHBV film. RT-PCR analysis ofthe proliferated cells on tissue culture polystyrene(TCPS) and porous and nonporous PHBV scaffoldsrevealed that the proliferated cells retained their osteo-blastic phenotype characteristics. Atomic absorption anal-

ysis was performed to measure the extent of calcium con-version by the cells grown on PHBV and TCPS. The cal-cium content of the culture media was used to indirectlymeasure the mineralization ability of the cells. The extentof calcium conversion by the cells was found to dependon the incubation time. Based on the results of the study,modified PHBV matrix seems to be a suitable matrix can-didate for bone tissue engineering application. � 2009Wiley Periodicals, Inc. J Biomed Mater Res 92A: 922–930,2010

Key words: scaffold; bone tissue engineering; PHBV; bio-degradable; UMR-106; BSP

INTRODUCTION

Many new polymers and polymer compositeshave been assayed for a broad range of applicationsincluding drug delivery,1,2 gene therapy,3 dental ma-terial,4 and tissue engineering.5–7 In particular, onepolymer that has drawn considerable attention forbone tissue formation is poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). PHBV is a natural bio-degradable copolymer produced in sufficient quan-tity by microorganisms such as Pseudoalteromonassp.8–16 By changing its composition, PHBV degrada-

tion and stiffness can be tuned to meet the require-ments for desired applications.17,18 When thehydroxyvalerate (HV) content in PHBV is 0%,Young’s modulus is 0.3 GPa; however, when the HVcontent in PHBV is 12%, Young’s modulus is 0.6GPa.19 In addition, PHBV has many useful biologicalproperties for clinical applications. PHBV, whenused as a suture, does not cause acute inflammation,necrosis, calcification of the fibrous capsule, or ma-lignant tumor formation within 1 year.20,21 PHBVmatrices sustain fibroblast cell proliferation whilemaintaining the structural integrity. PHBV has alsobeen noted to be a promising scaffold material forbone tissue formation.22

One approach to promote tissue formation onPHBV is to design the scaffold with improved bioac-tivity so that it can receive and respond to specificbiological signals, which direct and promote cellattachment, proliferation, differentiation, and tissueregeneration. There are several approaches toimprove PHBV scaffold bioactivity. Coating PHBVwith extracellular matrix proteins is a common prac-tice to enhance cell attachment.23 Treating PHBVwith oxygen plasma can also enhance cell attach-ment and proliferation.24 The microstructure of

*Present address: Department of Chemistry and Geol-ogy, Columbus State University, 4225 University Ave.,Columbus, GA 31907.Correspondence to: D. Raghavan; e-mail: draghavan@

howard.eduContract grant sponsor: NSF; contract grant number:

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

Material Command; contract grant number: DAMD 17-01-1-0268Contract grant sponsor: NIH; contract grant number:

S06GM 008016-35

� 2009 Wiley Periodicals, Inc.

porous scaffolds can also influence cell attachmentand infiltration as well as nutrient transport acrossthe polymer scaffold.25

Recently, we reported an approach for preparingmacroporous PHBV films and testing the biocompati-bility of PHBV scaffolds by initially leaching the saltfrom PHBV/salt film and coating the PHBV film withcollagen.26 UMR-106 cells were used as the osteoblastmodel system to evaluate the biocompatibility ofmodified PHBV. We demonstrated that UMR-106cells retained their osteoblastic phenotype characterwhen cultured on the engineered PHBV matrix. Inthis study, we have related PHBV film porosity andcollagen coating to UMR-106 cell proliferation. Inaddition, semiquantitative data about calcium conver-sion of the nutrient medium by cells grown on PHBVand tissue culture polystyrene (TCPS) have been pro-vided. RT-PCR analysis has been performed to pro-vide semiquantitative assessment of UMR-106 RNAexpression on TCPS and PHBV scaffolds.

MATERIALS AND METHODS

Materials

Poly(3-hydroxybutrate-co-3-hydroxyvalerate) (PHBV)containing 8 wt % HV, Coomassie Brilliant Blue G-250,95% ethanol, type I collagen (from calf skin), and sodiumchloride were purchased from Sigma-Aldrich (St. Louis,MO). The 6-well (No. 3516) and 96-well (No. 3596) TCPSwere purchased from Costar: Corning Incorporated(Corning, NY). Chloroform, filter paper (Cat No. 09-803-5F), and 85% (wt/vol) phosphoric acid were purchasedfrom Fisher Scientific (Hampton, NH). AeraSeal sterilesealing film was purchased from RPI Corp. (Mount Pros-pect, IL). Glyceraldehyde 3-phosphate dehydrogenase(GAPDH) and bone sialoprotein (BSP) DNA primer pairswere purchased from Sigma-Genosys (St. Louis, MO), and6% Tris-Borate-EDTA polyacrylamide (TBE-PAGE) gelswere obtained from Invitrogen (Carlsbad, CA). Also 53DNA loading solution, 103 TBE buffer, molecular biologygrade water, and Dulbecco’s modified essential medium(DMEM) were purchased from Quality Biological Inc. (Gai-thersburg, MD). DNA Ladder I was obtained from Gene-Choice Inc. (Frederick, MD). CellTiter 961 AQueous non-radioactive cell proliferation assay (MTS) with MTS andPMS stock solutions, SV Total RNA Isolation System, andAccess RT-PCR system were purchased from Promega(Madison, WI). Rat osteosarcoma UMR-106 (ATCC CRL-1661) cell line was purchased from American Type CultureCollection (Manassas, VA).

Preparation of PHBV scaffolds with salt leaching

A combination of solvent casting and solute leachingtechniques was used to prepare porous PHBV scaffolds.Briefly, 1.05 g of sieved sodium chloride (�150 lm) was

mixed with 0.7 g of PHBV powder manually. Hand mixedPHBV/salt mixture was dissolved in 7 mL of chloroformat 608C. The PHBV/salt solution was cast on a cleanedglass Petri dish. Solvent was allowed to evaporate over 24h at room temperature to form a film with a diameter of 6cm. To further remove solvent remnants in the film, vac-uum was applied for additional 24 h at room temperature.PHBV/NaCl composite film was then thoroughly washedwith distilled water at room temperature for several days.Wash solution was replaced by fresh water every 2 h forthe first 8 h and then 2–3 times a day to remove free chlo-ride ions. The washing step was continued until chlorideions were not detected in the leachate. This was verifiedby withdrawing an aliquot of the filtrate solution and test-ing it with 0.1N AgNO3 solution. The salt-leached PHBVscaffold was air- and vacuum-dried until a constant weightof the scaffold was reached and then stored in a desiccatorbefore use. For comparison, nonporous PHBV film wasprepared in an identical fashion by casting the solution,drying, and subjecting the film to air- and vacuum-drying.A portion of the porous and nonporous PHBV scaffoldswas saved for thermogravimetric characterization, and theremaining scaffolds were subjected to surface treatments.All films were modified as described in Ref. 26 except thefilms that were used for cell proliferation studies. Unlikeall other films, films used for cell proliferation studieswere not exposed to ozone treatment.

Collagen treatment of PHBV scaffolds

PHBV films were dipped into a type I collagen solution(1 mg/mL in 0.1 N acetic acid) for 24 h at 2–48C. The colla-gen-coated scaffolds were air-dried for 24 h at 2–48C. Gen-tle washing of the film was performed with serum-freeDMEM to remove remnants of acetic acid. The collagenadsorbed on the PHBV scaffold was measured via theBradford Method before and after adsorption.27 In addi-tion, we measured the collagen concentration after wash-ing the collagen adsorbed film for 20 h.

Briefly, 25 mg of Coomassie Brilliant Blue G-250 wasdissolved in 12.5 mL of 95% methanol. About 25 mL of85% (wt/vol) phosphoric acid was added, and the solutionwas diluted to 250 mL by deionized (DI) water. The rea-gent prepared was filtered through filter paper prior touse for collagen measurement.

The collagen-coated film was placed in a glass containeralong with 3 mL of DI water and 3 mL of prepared rea-gent. The reagent mixture and the film were in contact for10 min at room temperature. The film was retrieved, andthe absorbance of the solution was recorded at 595 nm.The amount of collagen absorbed on the PHBV film wasdetermined by relating the absorbance of solution beforeand after adsorption to the standard absorbance/originalconcentration curve.

Characterization of PHBV scaffolds:Thermogravimetric analysis

The salt fraction in the PHBV/NaCl composite film andporous PHBV film was quantified by performing thermo-

BIOLOGICAL RESPONSE OF OSTEOBLAST-LIKE UMR-106 CELLS 923

Journal of Biomedical Materials Research Part A

gravimetric analysis (TGA). Thermograms of the porousand composite PHBV film were obtained by heating 20 mgof the sample from 508C to 5508C at a constant rate of108C min21using TGA (PerkinElmer, Series 1) instrumentin a nitrogen atmosphere (40 mL/min).

Evaluation of cellular behaviors on the scaffold

Cell culture of UMR-106 cells

Osteoblast-like UMR-106 cells were cultured in DMEMsupplemented with 10% (vol/vol) heat-inactivated fetal bo-vine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin,and 100 lg/mL (wt/vol) streptomycin (Quality Biological,Inc.) at 378C in 5% CO2

26.

Cell proliferation on PHBV scaffolds

To measure the cell activity on PHBV scaffold, adherentosteoblast-like UMR-106 cells were cultured on severalPHBV films (with and without porosity as well as with andwithout collagen treatment). Because UMR cells exhibit dif-ferent attachment efficiencies on porous PHBV film vs.TCPS (1:3 PHBV to TCPS adherent cell ratio as measured at20 h incubation),15,26 care was taken to normalize and quan-tify the number of adherent cells on the various matrices atthe start of the proliferation study. About 3.0 3 104 UMR-106 osteoblast-like cells were seeded onto PHBV films, and1.0 3 104 UMR-106 osteoblast-like cells were seeded ontoTCPS, a positive control. All cells were cultured in serum-free DMEM to facilitate adhesion. The 96-well plates werecovered with sterile AeraSeal to promote gas exchange andincubated at 378C in 5% CO2. After 20 h, nonadherent cellswere removed via aspiration, and PHBV scaffolds with ad-herent cells were transferred to a new 96-well plate thatcontained DMEM supplemented with 10% fetal bovine se-rum. Similarly, nonadherent cells were removed via aspira-tion from TCPS well after 20 h incubation, and DMEM sup-plemented with 10% fetal bovine serum was added toTCPS with adherent cells. Each culture plate was coveredwith sterile AeraSeal and incubated at 378C in 5% CO2. TheMTS assay was used to measure proliferation in terms ofmetabolic activity28 between 4, 6, and/or 10 days.

Briefly, phenazine methosulfate (PMS) stock, the novel tet-razolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium, inner salt;MTS stock], and DMEM were mixed in the following volumeratio 1:20:84. Of this, 150 lL of the solution was added to thewells containing PHBV film or TCPS, and the 96-well platewas incubated at 378C for 2.5 h. Viable cells interact withMTS to form MTS formazan. Next, an aliquot (125 lL) waswithdrawn from the well, and the MTS formazan absorbancewas measured by a Dynatech MR5000 plate reader at 490nm. A similar method was utilized to establish a MTS stand-ard curve from a known number of UMR-106 cells.

Calcium conversion

Calcium conversion by the osteoblastic cells wasinduced by transferring the film with proliferated cells to a

well of a 96-well plate that contained mineralization me-dium [DMEM supplemented with 50 lg/mL L-ascorbicacid, 100 U/mL penicillin, 100 lg/mL streptomycin, 10%FBS, and 10 mM b-glycerophosphate (b-GP)]. Viable UMR-106 cells can hydrolyze organic phosphate to release inor-ganic phosphate ions, which can combine with Ca2þ ionsin solution to form a calcified apatite.29 We allowed cellsto grow in DMEM (proliferation medium) for a definedtime period followed by the transfer of cells to a minerali-zation medium for an additional time period. The nomen-clature 2d þ 10d imply 2 days proliferation followed by 10days in mineralization medium. When cells were grown inmineralization medium, care was taken to withdraw theold medium for calcium measurement and replace it withfresh medium every 2–3 days. All cell cultures were incu-bated at 378C with 5% CO2.

Atomic absorption spectroscopy was used to determinethe calcium content of the medium before and after cellseeding on TCPS and PHBV film. An aliquot, 50 lL, ofcell culture medium was mixed with 50 mL of DI water,and the absorbance of the solution was determined byusing an atomic absorption spectrometer (PerkinElmerAAS 800). The spectrometer was operated with 422.7-nmflame.30 Standard solutions containing known concentra-tions of calcium ions were prepared to establish absorb-ance–concentration calibration curve. The absorbance ofthe test solution was related to the calcium concentrationof the standard solution using the calibration curve. Blankswere run before, during, and after the measurement of testsolutions to address carry over effect. Triplicate measure-ments of each test solution were run to check the reprodu-cibility of the data.

RNA extraction and RT-PCR analysis

Collagen-coated porous PHBV scaffolds (thickness 340lm and diameter of 32 mm) and serum-free DMEM wereplaced into each well of a 6-well plate. About 1.4 3 106

cells were seeded onto the collagen-coated PHBV scaffolds,and 0.47 3 106 osteoblast-like UMR cells were seeded ontothe TCPS positive control. In a separate experiment, non-porous PHBV scaffolds (thickness 340 lm and 6 mm diam-eter) and serum-free DMEM were placed into wells of a96-well plate. About 3.9 3 105 cells were seeded onto non-porous PHBV, while 1.3 3 105 cells were seeded ontocontrol TCPS wells. As mentioned previously, the celladhesion efficiency was taken into consideration so thatsimilar number of cells would attach to TCPS and PHBVbefore the cell proliferation study was conducted. All cul-tures were incubated at 378C in 5% CO2. After 20 h, PHBVscaffolds with adherent cells were transferred to new wellsand then cultured with DMEM supplemented with 10% fe-tal bovine serum for 6 days. Total RNA was extractedfrom UMR-106 cells that were grown on PHBV scaffoldsand TCPS with the SV Total RNA Isolation System.The concentration of recovered RNA was determined bymeasuring the absorbance at 260 nm (A260) in a spectro-photometer.

RT-PCR analysis was performed to monitor geneexpression. DNA primers were chosen to study GAPDH (ahousekeeping gene) and bone sialoprotein (BSP) (a marker

924 LIU ET AL.


of the osteoblastic phenotype) levels in cells. The forwardprimer sequence for GAPDH was 50-ACT TTG TCA AGCTCA TTT CC-30 and the reverse primer sequence was 50-TGC AGC GAA CTT TAT TGA TG-30,31 while the forwardprimer sequence for BSP was 50-GAA ACG GTT TCC AGTCCA G-30, and the reverse primer sequence was 50-TGAAAC CCG TTC AGA AGG-30.32 One microliter of the iso-lated RNA solution was added to 49 lL of RT-PCR mastermixture.26 Also 1 lL of nuclease-free water (from theAccess RT-PCR system) was added to 49 lL of RT-PCRmaster mixture, and it served as a negative control. A one-step reverse transcriptase/DNA polymerase reaction wasperformed under the following conditions: 45 min at 458Cfor reverse transcription, 948C for 2 min to inactivate thereverse transcriptase, followed by 40 cycles of 30 s at 948Cfor melting, 1 min at 608C for annealing, and 2 min at688C for extension. The amplified cDNA product wasmaintained for an additional 7 min at 688C and thenstored at 48C. Ten microliters of cDNA solution was mixedwith 5 lL of 53 DNA loading solution. The combined so-lution was heated at 758C for 3 min in a water bath. Fif-teen microliters of each sample and 7 lL of the preparedDNA Ladder I (size maker) were resolved on a 6% TBE-PAGE gel in 13 TBE buffer at 200 V DC.

DNA Ladder I was used as an internal standard forcomparing the relative intensities of the transcriptsbetween individual gels, and QuantityOne software fromBio-Rad Laboratories was used to measure the relativeintensities of the amplified cDNA. Nuclease-free wateralong with RT-PCR master mixture served as a negativecontrol for RT-PCR analysis.

Statistical analysis

Reported data are present in the form of mean 6 stand-ard deviation (Mean 6 SD). A minimum of three measure-ments (n 5 3) was recorded for each sample set. Student’st-test was performed on the data, and the differencebetween measurements was considered statistically signifi-cant when p � 0.05. The statistical analysis of the data wasperformed using SigmaPlot statistical package software.

RESULTS AND DISCUSSION

Preparation and characterization of porousPHBV scaffolds

Porous PHBV scaffolds were made using solvent(i.e., chloroform) casting of PHBV/salt mixture andsolute (i.e., NaCl, �150 lm) leaching. Since the pres-ence of salt in the scaffold affects the biological func-tions of scaffold, PHBV/salt composite film waswashed thoroughly for an extended time period (7–10 days) until chloride ions were not visibly detecta-ble as a white AgCl precipitate upon addition ofAgNO3 to the leachate.

To establish that the sodium chloride was indeedremoved from the film, we performed TGA analysis

of the PHBV/salt film and the solute leachedPHBV/salt film. Figure 1 shows the TGA results ofPHBV/NaCl composite and porous PHBV scaffoldfilm. As a point of reference, the onset decomposi-tion temperature for PHBV (i.e., the temperaturewhen 5% mass loss occurs) is �2858C. As expected,we found that PHBV/NaCl composite (40/60 wt %)film lost �40% of the mass because of decompositionof PHBV at 3308C. No further mass loss wasobserved when the composite was heated up to�5508C, indicating that the remaining residue wasfree of polymer and it was primarily salt. In compar-ison, TGA of PHBV/NaCl composite film (40/60 wt%) and TGA of porous PHBV scaffold after saltleaching showed maximum mass loss at 3308C andnegligible mass loss beyond 3308C with little or noresidue. Thus, TGA data independently establishedthat sodium chloride was indeed washed out of theleached PHBV/salt film, and the leaching techniquethat was employed in our study removed the saltpresent in the composite film.

To establish that salt leaching leaves porous PHBVfilm, we collected optical images of the leached filmin our previous study.26 The optical micrographsshowed that pores were indeed present in the film,with pore dimension similar to the salt particle size(around 150 lm) used in the preparation of porousscaffold. The porous PHBV scaffold thus preparedwas used as the underlying scaffold for cell prolifer-ation study.

Effect of porous structure on cell proliferation

Cell proliferation on the scaffold is essential fortissue formation. Since, cells attached (as measuredwithin 20 h of incubation) at similar rates on both

Figure 1. TGA analysis of (a) PHBV/NaCl composite (b)porous PHBV scaffold film.



nonporous and porous PHBV film and at a higherrate on TCPS, the number of cells seeded on the var-ious matrices was normalized. To establish whetherthe presence of pores in the film can influence cellproliferation, osteoblast-like UMR-106 cells wereseeded on porous and nonporous PHBV films andthe MTS assay was performed at 4, 6, and 10 days.TCPS was included as a positive control. A standardcurve was generated by plotting MTS absorbanceversus cell number (as ascertained by hemocytome-ter and dilution), and a linear trend was observed.The MTS absorbance data of cells that proliferatedon PHBV and TCPS was compared against a stand-ard curve to determine the amount of cells that pro-liferated on porous and nonporous PHBV films.During the 10-day measurement, we noticed thatTCPS supported maximum cell proliferation fol-lowed by the porous scaffold, whereas nonporousscaffolds supported the least cell proliferation. Fig-ure 2 summarizes the cell proliferation results of po-rous PHBV, nonporous PHBV, and TCPS film. After10 days, the number of the cells on the porous scaf-fold was nearly six times that of the cells on the non-porous scaffold. Moreover, MTS data suggest thatcells seeded on porous PHBV exhibit nearly threedoublings between days 1 and 10, whereas cellsseeded on nonporous PHBV exhibit only one dou-bling over the same period. The ability of porousPHBV scaffold to promote cell proliferation wasdirectly attributed to the existence of porous struc-ture in the film.33 Similar observation of enhancedcell proliferation on porous PLLA scaffold has beenreported.34 A porous scaffold presents more surfacearea for cell attachment and proliferation, and it ena-bles nutrient transfer and cell infiltration into thebulk film. These observations were found to be ingeneral agreement with the results reported by Koseet al.,24,35 who noticed that primary rat stromal/osteoblastic cells proliferated better on porousplasma-treated PHBV foams.

Effect of collagen dip-coating on cell proliferation

Type I collagen has been commonly used to coatscaffold surfaces because it is readily available and itcan enhance the attachment of osteoblast-like cells tothe polymeric scaffold.36,37 Porous and nonporousPHBV films were dip-coated with type I collagen fol-lowed by gentle washing.38,39 By Bradford method,we established that the collagen density on porousPHBV film to be 11 lg/sample. About 3 3 104

UMR-106 cells were seeded onto PHBV films andallowed to incubate for 4, 6, and 10 days for prolifer-ation studies as described earlier.

Figure 3 shows UMR cell proliferation on porousPHBV films with and without collagen. We noticedthat the cells proliferated to a greater extent on thecollagen-coated porous PHBV scaffold than on thenoncoated porous PHBV scaffold. Student’s t-testwas applied to ascertain whether the means of cellproliferation of the paired groups at different timepoints are statistically significant. A confidence levelabove 95% was considered statistically significant.Our results indicated that collagen coating of scaf-fold further enhances cell proliferation on the matrix.However, porosity has a bigger influence on cell pro-liferation than collagen dip-coating. For example, af-ter 6 days, the cell density increased nearly 4 timesfor cells cultivated on porous membranes lackingcollagen, whereas it increased nearly 6 times for cellscultivated on porous membranes with collagen.

Calcium measurement

In our previous study,26 it was demonstrated thatUMR-106 cells when grown on the PHBV matrixwere able to convert calcium (present in themedia) to a mineral phase. The calcium conversionin the media was qualitatively followed by alizarin

Figure 2. Cell proliferation on porous and nonporousPHBV scaffold. *p > 0.05, **p < 0.05 (t-test).

Figure 3. Cell proliferation on porous PHBV scaffoldswith and without collagen coating. *p > 0.1, **p < 0.05,***p < 0.1 (t-test).

926 LIU ET AL.


red technique. In this study, we have made anattempt to quantify the decrease in calcium ion con-centration of the medium as a function of incubationtime. Calcium conversion was induced by transfer-ring the cells attached to a matrix to a new growthmedium supplemented with b-GP. It must be notedthat whenever the cells were allowed to grow inDMEM medium supplemented with b-GP for morethan 2 days, the medium was replaced with freshmedium every 2–3 days, and the spent DMEM me-dium with b-GP was saved for calcium measure-ment. If the cells were cultured in the DMEMmedium supplemented with b-GP for 7 days, themedium was replaced at least three times, and thespent media were saved for calcium measurement.The calcium concentration of the spent and freshmedium was measured by atomic absorption spec-trometry (AAS). By measuring the calcium contentof the individual spent media and knowing the totalcalcium content in the original media, the amount ofcalcium converted in the individual spent mediawas obtained. Also, the amount of calcium convertedduring the entire incubation period was obtained byadding the amount of the calcium converted in theindividual spent media.

Figure 4 is a plot of the estimated calcium con-verted in the medium as a function of incubationtime. We noticed that when cells are allowed to growfor identical incubation time period on PHBV andTCPS, the extent of calcium conversion by the cellswas found to be far more on TCPS than on PHBVmatrix. This may be a result of slower proliferationrate of the cells on PHBV matrix than on TCPS or dif-ferences in osteogenic differentiation potential.40,41

The amount of calcium converted by the cellsgrown on a matrix was found to depend on the

incubation period. We observed minimal calciumconversion of the medium when the matrix withattached cells was incubated for 10 days in DMEMand 2 days in DMEM supplemented with b-GP.Conversely, calcium conversion of the medium wasfound to be maximum when the PHBV matrix withattached cells was incubated for 20 h in DMEM and12 days in DMEM with b-GP. Our findings are ingeneral agreement with the observations reported byGregory et al.42 and Donzelli et al.,43 where alizarinred-stained calcium deposits was noticed on the sub-strate. When the matrix with attached cells isallowed to grow in growth medium supplementedwith organic phosphate for a longer time, a greateramount of stained deposit is formed.

RT-PCR analysis

In addition to measuring the calcium conversionby cells proliferating on the matrix, it is desirable toinvestigate whether the phenotypic characteristics ofthe cells will change because of proliferation on

Figure 4. Calcium converted by the cells that proliferatedon the matrix as a function of incubation (for example, 2dþ 10d represents incubation of attached cell in DMEM me-dium for 2 days followed by incubation in DMEM me-dium supplemented with b-GP for 10 days).

Figure 5. RT-PCR analysis of UMR-106 cell cultures.RNA was extracted from the cell cultures at 0 and 6 daysafter the addition of DMEM media. These extracts weresubjected to RT-PCR analysis with rat-specific primers forglyceraldehyde-3-phosphate dehydrogenase [GAPDH, Fig-ure 5(a)] and bone sialoprotein [BSP, Figure 5(b)]. PCRproducts were separated on a 6 % TBE-PAGE gel, stainedwith ethidium bromide and visualized over a UV light(lane A1: PHBV 20h; lane B1: TCPS 20h; lane A2: PHBV6d; lane B2: TCPS 6d; lane C: negative control).



PHBV matrix. UMR-106 cells obtained from rat arecommonly used as a model bone cell type to evalu-ate the cell viability on engineered scaffold for bonetissue applications.29

In our previous study, two different markers, glyc-eraldehyde 3-phosphate dehydrogenase (GAPDH) (ahousekeeping gene) and bone sialoprotein (BSP) (amarker of the osteoblastic phenotype), were exam-ined after the UMR-106 cells were cultivated onTCPS and porous PHBV scaffold for 10 days. RT-PCR analysis of RNA extracted from the cells grownon TCPS or PHBV matrix gave expected cDNA frag-ments of 267 bp and 564 bp that correspond to theGAPDH and the BSP transcripts, respectively.26 Wealso conducted RT-PCR analysis of RNA extractedfrom UMR-106 cells grown on nonporous PHBVfilm and observed cDNA fragments of 267 bp and564 bp suggesting that UMR cells retained osteoblas-tic phenotype expression, regardless of the nature ofPHBV film (unpublished data). Since a predominantamount of cell proliferation work was performed onporous PHBV film, we limited our semiquantitativeRT-PCR analysis to porous PHBV film.

To obtain relative value of the two marker bandsfor the cell population grown on TCPS and porousPHBV, the intensity of negative control was sub-tracted from the measured intensity of the twomarker bands. As shown in Figure 5(a,b), the bandintensity of the two markers was found to increaseafter 6 days of incubation. For example, on day 6,the GAPDH cDNA level was 7 times more intensethan the GAPDH cDNA observed on initial cell cul-ture. Similarly, on day 6, the BSP cDNA level wasfound to be 8 times greater than the BSP intensitylevel observed for the initial cell culture. An increasein the BSP message level is consistent with our ear-

lier findings of alizarin red staining data26 and thecalcium conversion data reported in this study. Fur-thermore, our findings are in general agreementwith observations reported by Kose et al., wherealkaline phosphatase activity data were used to sup-port the suitability of PHBV matrix for bone tissueapplications.35

A comparison of the relative intensity of BSP nor-malized to GAPDH (internal standard) showed thatcells grown on TCPS and PHBV remained relativelyconstant during the measurement period (Fig. 6).Student’s t-test was applied to ascertain whether themean relative intensities of BSP normalized toGAPDH are statistically significant. Since the relativeintensity of BSP normalized to GAPDH (the internalstandard) remained relatively constant and the num-ber of cells increased with time, it suggests that onan average, the UMR-106 cells are continuing to ex-hibit osteoblastic (BSP) phenotypic expression.

CONCLUSIONS

Biocompatibility is an important criterion to beevaluated for consideration of polymer scaffold fortissue engineering applications. Our study showedcollagen immobilization on porous PHBV filmresulted in improved biocompatibility, as evidencedby enhanced osteoblast-like cell attachment and pro-liferation. More importantly, we found that osteo-blast-like cells grown on the PHBV film retainedtheir bone mineralization ability, which was con-firmed by calcium conversion. Osteoblast-like cellsretained their osteoblastic phenotype characteristics,as ascertained by intensity of characteristic markers(GAPDH and BSP gene expression). The relative in-tensity of BSP normalized to GAPDH (internalstandard) showed that cells grown on PHBV matrixremained relatively constant during the measure-ment period. These results suggest that the engi-neered PHBV scaffolds with improved biocompati-bility warrant their further testing for possible use inbone tissue engineering applications.

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