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BioPEGylation of Polyhydroxyalkanoates: Influence on Properties and Satellite-Stem Cell Cycle

BioPEGylation of Polyhydroxyalkanoates: Influence on Properties and Satellite-Stem Cell Cycle
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  BioPEGylation of Polyhydroxyalkanoates: Influence onProperties and Satellite-Stem Cell Cycle Helder Marc¸al, † Nico S. Wanandy, † Vorapat Sanguanchaipaiwong, † Catherine E. Woolnough, † Antonio Lauto, † Stephen M. Mahler, ‡ and L. John R. Foster* ,† Bio/Polymer Research Group and Centre for Advanced Macromolecular Design, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW2052, Australia, and Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Qld 4072, Australia Received April 17, 2008; Revised Manuscript Received July 24, 2008  The addition of poly(ethylene glycol), PEG, to bioprocessing systems producing polyhydroxyalkanoates (PHAs),has been reported as a means of their molecular weight control and can also support bioPEGylation, resulting inhybrids with amphiphillic properties. However, the study of such natural-synthetic hybrids of PHA- b -PEG is stillin its infancy. In this study, we report the influence of bioPEGylation of polyhydroxyoctanoate (PHO) on itsphysiochemical, material, and biological properties. Consistent with previous studies, bioPEGylation with diethyleneglycol (DEG) showed a significant reduction in PHA molecular weight (57%). In comparison to solvent castfilms of PHO, PHO- b -DEG films possessed a noticeable X-ray diffraction peak at 9.82 °  and increased Young’smodulus of 11 Gpa (83%). Potential biocompatibility was investigated by measuring the early phase of apoptosisin myoblastic satellite-stem cells (C2C12). Comparative analysis of cell proliferation and progression in the presenceof the mcl-PHA and its hybrid showed that the latter induced significant cell cycle progression: the first time abiomaterial has been shown to do so. Microtopographies of the film surfaces demonstrated that these differenceswere not due to changes in surface morphology; both polymers possessed average surface rugosities of 1.4 ( 0.2  µ m. However, a slight decrease in surface hydrophobicity (3.5  (  0.9 ° ) due to the hydrophilic DEG may haveexerted an influence. The results support the further study of bioPEGylated PHAs as potential biomaterials in thefield of tissue engineering. Introduction Biomaterials used in tissue engineering applications serve assupport scaffolds and adhesive substrates for cells during in vitroculture and subsequent to implantation. 1 The design andselection of scaffolding biomaterials can significantly influencethe development of engineered tissues. Various biopolymershave emerged as potential candidates supporting cell adhesion,proliferation and differentiated function. 2 - 4 A number of biopolyesters from the family of polyhydroxyalkanoates (PHAs)have been investigated for such roles.PHAs are synthesized by a wide variety of microorganismsunder unbalanced growth conditions and serve a carbon andenergy storage function. 5 To date, over 105 different monomericstructures have been identified as components of PHAs, but onlyseveral have been investigated for their potential as tissueengineering materials. 6,7 Of these, the focus has been on PHAscomprised of monomers with relatively short chain lengths (4 - 6carbon units, scl-PHA), mainly poly(hydroxybutyrate) (PHB),and its copolymers. 7 The PHB homopolymer consists of hydroxybutyric acid (HBA) monomers with the microbialmonomer chemically identical to mammalian HBA producedas one of the ketone bodies during prolonged starvation andunder diabetic conditions. 8 Recently, Chen and co-workers havespeculated that 3-HBA supports tissue regeneration by prevent-ing cell apoptosis. 9,10 In contrast to scl-PHAs, studies on PHAswith monomers of medium chain lengths (6 - 16 carbon units,mcl-PHA) are limited and restricted to the terpolymer poly(hy-droxyoctanoate) (PHO). 7 PHAs and their composites have also been used to producea variety of medical devices, from sutures and bone plates tovein valves, various wound dressings, and tissue regenerationdevices (for a detailed review, see ref 7). In vitro and in vivostudies using PHA-based biomaterials have shown equivocaldifferences in biocompatibility and biodegradability. 11,12 It hasbeen suggested that differences in biocompatibility betweenPHAs may be due, in part, to variations in physical andmorphological properties such as microtopography and surfacehydrophobicity. 13,14 Thus, a further understanding of thecomplex interface events that occur between cells and abiomaterial surface is necessary when engineering biopolymers.This is due to the influence that biopolymers induce on cellularresponses at the material interface. 15 Surface chemistry, inparticular, is known to affect cell function and adhesion. 16 It istherefore desirable to engineer polymers that are designed toincorporate modifications at the material surface that are suitablefor their intended applications.Molecular mass and monomeric composition of PHAs and,by extension, their material properties, including surfacechemistry, can be controlled through the addition of poly(eth-ylene glycol) (PEG). 17 Furthermore, PEGs with molecularweights below approximately 600 can act as chain terminatingagents resulting in the biosynthesis of natural-synthetic hybridsof PEGylated PHAs, that is, “bioPEGylation”. 17 The majorityof research in this field has focused on the PEG modulatedcontrol of PHA biosynthesis, little has been done to considerPHA- b -PEG hybrids as biomaterials. 17 We have recentlydemonstrated that the hybrid polymer chains can be induced to * To whom correspondence should be addressed. Tel.: ( + 61)2-9385-2054. Fax: ( + 61)2-9385-1483. E-mail: † University of New South Wales. ‡ University of Queensland. Biomacromolecules   2008,  9,  2719–2726  2719 10.1021/bm800418e CCC: $40.75  ©  2008 American Chemical SocietyPublished on Web 08/29/2008  exhibit a degree of self-assembly for the fabrication of disor-dered microporous films that have potential for cell immobili-zation. 17,18 In this study, we compare the physiochemical and materialproperties of PHO with its bioPEGylated hybrid “end-capped”with diethylene glycol (DEG as PEG106). Furthermore, weassessed the biocompatibility of PHO and PHO- b -DEG withmyoblastic satellite-stem cells derived from skeletal muscle. Thispreliminary test for biocompatibility was determined by analyz-ing the early stages of apoptotic activation and investigatingthe influence of the polymers on cell cycle progression. To thebest of our knowledge, this is the first time that the influenceof the biomaterial on satellite-stem cell cycle has beeninvestigated.The seeding of biopolymeric scaffolds with stem cells forthe generation of tissue-engineered products requires two cellularprocesses to occur concurrently. Cells must spread and populatethe scaffold through cell proliferation, whereby cells cyclethrough phases classified as “G0/G1” (resting), “S” (DNAsynthesis), “M” (mitosis), and “G2” (gap between S and M).Typically, it is the G0/G1 phase that is shortened when cellsmove from a nonproliferative to a proliferative state, while S,G2 and M remain relatively constant. Concurrently, cellsundergo differentiation whereby cells are programmed to fulfillspecific functions. Skeletal muscle-derived satellite-stem cellsare recognized to provide repair and regeneration following localinjury of muscle fibers. 19 In undamaged adult tissues, satellite-stem cells are quiescent myocyte cells that are usually detected just beneath the basal lamina. 20 However, following injury andduring muscle regeneration, the satellite-stem cells leavequiescence and re-enter the cell cycle to proliferate as myoblasticcells to replenish the tissue. Ultimately, they reach terminaldifferentiation and fuse together to form myotubes and myofi-bers. 21 Manipulation of their growth using biomaterials istherefore of considerable interest, as is the suitability of suchbiomaterials in tissue engineering. Materials and Methods Materials.  Mammalian cell growth medium and fetal bovine serum(FBS) were purchased from Gibco-Invitrogen (Sydney, Australia). Stemcells (C2C12) were routinely cultured in Dulbecco’s Modified Eagle’sMedium (DMEM/F12) supplemented with 10% FBS. Trypsin waspurchased from Sigma-Aldrich (St. Louis, MO) and cell arresting agentsaphidicolin and nocodazole were purchased from Sigma-Aldrich(Sydney, Australia). Stock solutions of aphidicolin and nocodazole wereprepared in dimethyl sulfoxide (DMSO) and stored at  - 20  ° C. A kitto determine endotoxin levels was purchased from Sigma (Sydney,Australia). Octanoic acid, DEG (as PEG106), and microbially producedPHB were purchased from Sigma-Aldrich (Sydney, Australia). All otherchemicals were obtained from APS Chemicals (Seven Hills, Australia)and were of analytical grade with minimum 98% purity. Subculturing of Satellite-Stem Cells.  Adherent cells cultivated insterile T25 tissue flasks were subcultured in DMEM - 10% FBS mediumwhere they attained approximately 90% confluence. The spent mediawas aspirated, rinsed twice with PBS, and 2 mL of trypsin (0.12%)was added to cover the cells, which were then incubated at 37  ° C for2 min. Cells were subsequently dislodged and transferred to sterilecentrifuge tubes before centrifuging (10 min at 300 × g ). Supernatantswere decanted, and cell pellets were resuspended in 10 mL of freshmedium. Of this, 1 mL aliquots were transferred to new T-flasks, eachcontaining 9 mL of fresh medium (i.e., 1 in 10 dilutions). Cells wereimmediately incubated at 37  ° C after gassing with 5% CO 2  forapproximately 10 s. Polymer Production.  PHO and PHO- b -DEG were produced throughsimultaneous cultivations of   Pseudomonas oleo V orans  (ATCC 29347)using the same inoculum, as described by Foster et al. 18 Octanoic acidwas used as a carbon source and 2% (w/v) DEG was added for theproduction of the bioPEGylated hybrid. Polymer samples were extractedinto chloroform from the lyophilised biomass and precipitated into coldmethanol. The samples were subsequently purified through a series of chloroform solvation and methanol precipitation cycles. 22 Purificationwas established by measuring the endotoxin content for each cycle. Table 1.  Summary of Physiochemical, Material, and BiologicalProperties of PHO- b  -DEG and its Solvent Cast Films Compared toPHOproperty PHOPHO- b  -DEGmolecular weight ( × 10 3 ) 235 100molecular weight number ( × 10 3 ) 142 77polydispersity index 1.65 1.30density (g/cm 3 ) 1.05 0.99melting point ( ° C) 55 ( ( 1.1) 55 ( ( 0.8)glass transition temperature ( ° C)  - 32 ( ( 1.4)  - 31 ( ( 1.1)enthalpy of fusion (J/g) 31 ( ( 3.5) 34 ( ( 2.7)crystallinity (%) 38 ( ( 3.4) 34 ( ( 2.9)Young’s modulus (Gpa) 6 ( ( 0.3) 11 ( ( 0.4)tensile strength (Mpa) 7 ( ( 0.7) 6 ( ( 0.7)extension to break (%) 580 ( ( 9.4) 540 ( ( 8.3)water contact angle ( ° ) 73.8 ( ( 0.9) 70.3 ( ( 0.7)Surface Tension (mN/m) 40 ( ( 0.5) 42 ( ( 0.4)surface tension (mN/m)  γ s1  17 ( ( 0.4) 19 ( ( 0.2)average surface roughness (  µ m) 1.5 ( ( 0.2) 1.3 ( ( 0.2) Figure 1.  Graph showing change in molar volume with increasingmole fraction for PHO ( - O - ) and PHO- b  -DEG (grey circle); PHB( - b - ) of known density used as a control. Figure 2.  DSC thermograms of (a) PHO and (b) PHO- b  -DEG asproduced using  P. oleovorans  . 2720  Biomacromolecules, Vol. 9, No. 10, 2008   Marc¸al et al.  Polymer compositions and bioPEGylation were confirmed using gaschromatography (GC) and 2D nuclear magnetic resonance spectroscopy( 1 H - 1 H COSY and  1 H - 13 C HSQC), as described in detail previously. 18 Briefly, polymer samples were dissolved in deuterated chloroform ( ∼ 4mg/mL) and then examined using a Bruker DMX600 (600.13 MHzfor  1 H and 150.92 MHz for  13 C).  1 H spectra were recorded with apulse width of 4.5 ms (458 pulse), a spectral width of 6.6 kHz, anacquisition time of 2.5 s, and a relaxation delay of 6 s. Between 64and 256 scans for the required signal-to-noise correction were collectedand all scans were referenced internally to chloroform (7.26 ppm withrespect to tetramethylsilane).Samples of purified PHO and PHO- b -DEG were dissolved inanalytical-grade chloroform and fabricated into thin films by castinginto clean, dry, sterile glass Petri dishes, dried for 48 h in a vacuumdesiccator and removed from the dishes before allowing to stand foran additional 48 h until their weights had atmospherically equilibrated.The films were then aged for an additional three weeks to enable theircrystallinity to reach equilibrium. For standardization purposes allmicroscopic imaging and cell cultivation was performed on film sidesthat were cast in contact to the clean glass. Polymer Characterization.  The molecular mass properties of thepolymers were determined by gel permeation chromatography usingan LC-10ATVP Shimadzu solvent delivery system combined with aSIL-10ADVP Shimadzu autoinjector possessing a stepwise injectioncontrol and a column set consisting of a PL 5.0 mm bead size guardcolumn and a set of 3 - 5.0 mm PL linear columns (103, 104, 105 Å)kept at a constant 40  ° C inside a CTO-10AC VP Shimadzu ColumnOven and an RID-10A Shimadzu refractive index detector. Sampleswere analyzed in a continuous phase of tetrahydrofuran (THF, 1 mL/ min) and values were calculated from a calibration curve of polystyrenestandards with low polydispersity ( < 1.1). 18 Thermal properties of the polymers were investigated using dif-ferential scanning calorimetry (Perkin-Elmer Model DSC-7). Samples(7 - 10 mg) were heated at the rate of 20  ° C/min from 80 to 127  ° C. 18 The curves obtained were used to calculate the glass transitiontemperatures ( T  g ), melting point ( T  m ) and fusion enthalpies ( ∆  H  f  ) withmeans of five samples determined. X-ray diffraction analysis of solventcast films was performed using a Siemens D-5000 diffractometer (30kV, 30 mA) with Monocrom Ka radiation (  λ  )  1.5418 Å).Polymer densities were determined using a calibrated Anton PaarDMA5000 density meter (Anton Paar, Graaz, Austria). Polymer samplesbetween 1 and 30 mg/mL were dissolved in analytical grade chloroformand analyzed at 20  ° C and 25% rH. A mean of 10 readings perconcentration was taken and expressed graphically as molar volume( V  m ) against mole fraction. Mole fractions of copolymers and hybridswere calculated based on their complete composition (i.e., PHO: C6,C8, C10 monomeric units). Extrapolation of linear curves fitted to thedata were used to determine the calculated bulk density for thepolymers. Samples of PHB with a reported density between 1.21 and1.25 g/cm 3 were used as a control. Material Properties of Polymer Films.  An Instron Mini55 tensi-ometer (Instron, MA) was used to measure the material properties forsolvent cast films of the mcl-PHA and its hybrid. Samples of PHO( × 25) and PHO- b -DEG ( × 11) were clamped to the calibrated tensi-ometer with pneumatic grips that gradually separated at a rate of 22mm/min until failure, the maximum load and extension to break weremonitored, and the tensile stress and Young’s modulus were statisticallydetermined. Surface Characterization of Polymer Films.  The surface mor-phologies of solvent cast films of PHO and PHO- b -DEG visualizedusing scanning electron microscopy while their microtopographies weremapped using the reflection mode of a confocal scanning lasermicroscope (CSLM, Leica model TCS-SP, Germany; excitation 458nm, emission 440 - 470 nm). Multiple images through the  z -plane wererecorded (step size  )  0.5  µ m). The average surface roughnesses (  R a )for the films were calculated from these images using ImageJ computersoftware (National Institute of Health, U.S.A.). A total of 10 imageswere taken and the average  R a  values were calculated. 23 The surface hydrophobicities of polymer films were determined usinga Rame´ Hart contact angle goniometer, (CAG, National ResearchLaboratories model 100-00, U.S.A.). Deionised sterile water (3  µ L)were placed onto the polymer film surfaces and an average of 10readings for each side recorded. These readings were then used todetermine the average surface hydrophobicity. Polymer Film Biocompatibility.  Cell growth and viability weredetermined using the trypan blue exclusion method. 24 Briefly, 100  µ Lsamples were routinely collected and subjected to trypan blue dye (finaldilution of  × 2, unless otherwise specified). A sterile pipet was used togently produce a homogeneous suspension before analysis using ahemocytometer (Neubauer, Weber, England). Viable cell concentrationsand percentage viable cells were calculated in accordance with “CurrentProtocols in Molecular Biology”. 24 Cells were cultured in DMEM/F12 containing 10% FBS on glasstissue culture dishes coated with films of PHO and PHO- b -DEG andgrown for 5 days with a working volume of 2 mL. Controls in theabsence of the polymers were simultaneously conducted from the sameinoculum. Additional controls were similarly conducted to validate theacquired data. Controls for DNA content were as follows: (1) cells in10% FBS, (2) serum-deprived cells, (3) cells synchronized for 24 h inmedium containing 10% FBS and 1  µ g mL - 1 aphidicolin, and (4) 2  µ M nocodazole. During harvesting, cells that were subjected to a DNAcontent assay were centrifuged at 300  g  and washed once in PBS beforebeing resuspended with cell cycle staining buffer at concentration 1 × 10 6 cells mL - 1 and incubated on ice for 30 min before acquisitionusing flow cytometry. Two sets of controls for apoptosis detection werealso conducted by employing cells that were subjected to 10  µ Mcampthotecin for 6 h as well as 2  µ M nocodazole for 24 h. Controlsfor apoptosis were performed in the presence of 10% FBS, whilenegative healthy controls were also conducted using the same conditionswith the absence of apoptosis inducing agents.Apoptotic indices associated with the externalisation of phosphoti-dylserine (PS) was assayed with FITC-conjugates Annexin-V apoptosiskit and counter stained with propidium iodide (PI) included in this kit(BD Bioscience, Pharmingen, U.S.A.). Staining was conducted inaccordance with the manufacturer’s protocol. Prior to analysis, cellswere harvested and washed once in PBS. FITC and PI molecules werevisualized using a Becton and Dickson FACS Calibur Flow Cytometer(San Jose´, U.S.A.), equipped with a 488 nm laser. Bivariate forwardand side-angle scatter gating were used to identify homogeneous Figure 3.  Microscopic images of myoblastic satellite-stem cellscultivated on films of PHO- b  -DEG (a) and PHO (b); bar  )  200  µ m. BioPEGylation of Polyhydroxyalkanoates  Biomacromolecules, Vol. 9, No. 10, 2008   2721  populations while excluding cellular debris and dead cells. FITC andPI fluorescence’s were visualized using 530/30- and 585/42-bandpassfilters, respectively (10000 events for Annexin-V and 50000 eventsfor a DNA content histogram were collected). Photomultiplier (PMT)voltages were adjusted to ensure that autofluorescence associated withnonapoptotic samples described a Gaussian distribution within the firsttwo log-decade on univariate histograms. Analysis was performed usingCytomation Summit v3.1 software (Cytomation, Fort Collins, CO).The PI staining solution was prepared by mixing 0.1% (v/v) TritonX-100, 0.1% (w/v) BSA, 40  µ g mL - 1 PI, and 10  µ g mL - 1 RNase Ain PBS. Cells were extracted through centrifugation at 300  g , rinsedonce in PBS, and centrifuged again. Cells were then resuspended,transferred to FACS tubes, and incubated in the staining solution for30 min on ice. PI fluorescence intensities were deconvulated usingModFit (Verity Software House, Inc., Topsham, ME) to resolve cellcycle distributions. For statistical accuracy means of experimental datawas accumulated from three repeat experiments with means of 10samples each. Statistical Analysis.  All data was statistically evaluated using thetwo-way ANOVA analysis and Bonferroni post-test (significance level )  0.05). Results and Discussion Polymer Properties.  GC and NMR analysis of the two PHAsproduced from the bioprocessing systems using octanoic acidas carbon source and octanoic acid with 2% (w/w) DEGconfirmed their composition as PHO and PHO- b -DEG, as wehave previously reported. 18,22 In this study, the PHO producedwas a mcl-PHA heteropolymer composed of monomeric units Figure 4.  Histograms of populations of cells with DNA exhibiting G0/G1, G2-M and S growth phases. Positive controls for (a) G0/G1 phasethrough serum deprivation, (b) S phase through addition of aphidicolin, (c) G2/M phase through addition of Nocodazole, (d) asynchronousgrowth, (e) PHO, and (f) PHO- b  -DEG. 2722  Biomacromolecules, Vol. 9, No. 10, 2008   Marc¸al et al.  with 6 (11.2), 8 (84.5), and 10 (4.3 mol %) carbons. Additionof DEG to the cultivation of   P. oleo V orans  for PHO productionresulted in a natural-synthetic block copolymer hybrid with adifferent monomeric composition of C6 (4.3), C8 (95.0), andC10 (0.7 mol %). Furthermore, peak integration ratios from  1 HNMR spectra, 2D NMR analysis and other analyses confirmedthe absence of a polymer mixture and no unbound DEGunits. 18,22 Consistent with previous reports, bioPEGylation of PHO withDEG resulted in a significant reduction in the weight averagemolecular weight (  M  w ) and number average molecular weight(  M  n ) when compared to PHO (57 and 54%, respectively), anda change in its polydispersity from 1.65 to 1.30 (Table 1). Thus, P. oleo V orans  was utilized to produce a natural-synthetic hybridcopolymer of PHO- b -DEG, which had a noticeably differentmonomeric composition and a significantly reduced molecularmass when compared to its nonhybridized counterpart, PHO.While these changes had no apparent effect on the molecularconformation of the polymer chains in solution, as determinedusing small angle neutron scattering (SANS), 25 bioPEGylationhad a significant influence on polymer density. Figure 1 showsthe noticeable differences in molar volume, with mole fractionfor PHO and PHO- b -DEG giving densities of 1.05 and 0.99g/cm 3 , respectively. To the best of our knowledge, there are noreports on the density of PHO and, consequently, we used PHB,with densities reported between 1.21 and 1.25 g/cm 3 forconfirmation of experimental accuracy (Figure 1, Table 1). 26 Despite the changes in its molecular properties, PHO- b -DEGshowed no significant differences in its thermal properties whencompared to PHO. Both polymers had melting points and glasstransition temperatures around 55 and  - 31.5  ° C, respectively(Table 1). However, the enthalpy profile for PHO- b -DEGexhibited an additional noticeable peak consistent with wateraround - 7  ° C and suggests that the hydrophilic DEG group inthe hybrid may have adsorbed water more readily than PHO(Figure 2). This suggestion is supported by Lee et al. whoreported increased hydrophilicity with side-chain hydroxylationin mcl-PHAs. 24 Similarly, solvent cast films of PHO- b -DEGshowed an increase in hydrophilicity compared to PHO (seePolymer Film Surface Properties).While the biocompatibility of PHAs has been established,toxicological investigations of these biopolymers show that theremay be inflammatory reactions which may be partly due tofailure of the purification process. 27 Commercially available scl-PHAs are reported to contain pyrogens in the form of li-popolysaccharide complexes from cell membranes. 27,28 Milleret al. report such endotoxin levels in commercial PHAs in excessof 100 U; in the studies reported here, purification of PHO andPHO- b -DEG proceeded through a cycle of dissolution inchloroform and precipitation in cold methanol. Endotoxin levelswere monitored during this purification and, after four cycles,had decreased to below 10 U, a level deemed suitable forbiomedical studies. 28,29 Satellite-Stem Cell Cycle.  Assessment of PHA biocompat-ibility has been shown in vitro with a variety of cell linesincluding chondrocytes, fibroblasts, and macrophages. 7 How-ever, in addition to biocompatibility, the influence of thesebiopolymers on cell cycling is also important in their consid-eration as biomaterials for tissue engineering. In tissue engineer-ing, stem cells spread and populate through a polymeric scaffold.During this process, the cells cycle through phases of quiescence(G0/G1), DNA replication (S), rest (G2), and finally, celldivision (mitosis, M). In the studies reported here, myoblasticsatellite-stem cells (C2C12) were cultivated on films of PHOand its natural-synthetic hybrid of PHO- b -DEG.Growth of stem cells on both the mcl-PHA and its bioPEGy-lated films was demonstrated with no noticeable differences inmorphology (Figure 3). Furthermore the DNA content of C2C12cells cultivated on PHO and PHO- b -DEG did not show anysignificant cell cycle aberrations (Figure 4). A qualitativeanalysis of the histograms in Figure 4 clearly show that thestem cells cycled through the G0/G1, S and G2, M phases.Positive controls using aphidicolin and nocodazole were usedto induce cells to enter S (DNA sysnthesis) and G2/M phases(gap and mitosis), respectively, while conditions of serumdeprivation were used to maintain cells in the G0/G1 quiescentphase. In comparison to these controls, cells cultivated on thePHO and PHO- b -DEG films showed no significant inhibitionsin their growth. However, statistical analysis of the data suggestssignificant differences in cell population densities when culti-vated on PHO- b -DEG.Cells cultivated in the presence of aphidicolin and nocodazole(controls) induced entry into the S and G2/M phases, respec-tively. These results were statistically different (  p < 0.01) whencompared to those for the control without biomaterials. Simi-larly, cell populations cultivated with serum deprivation werealso different, verifying the validity of the cultivation protocol.While the PHO film had a similar population in the G0/G1(quiescent or “resting”) phase as those grown in the absence of any biomaterials, that is, asynchronous growth ( ∼ 54%), thepopulation density in the S phase appeared slightly reduced witha greater proportion of cells found in the G2-M phase ( + 3.2%,Figure 4d,e). However, these differences were statisticallyinsignificant and, thus, the PHO did not alter satellite-stem cellcycling when cultivated under these conditions. In contrast,bioPEGylation apparently continued the insignificant change incell cycle trend observed for PHO, with even less cellspossessing DNA in the S phase ( - 10.6%, Figure 4d - f). Thus,for PHO- b -DEG, there were statistically significant differencesfor cell populations in the G2/M (  p  <  0.1) and S (  p  <  0.001)phases, when compared to the same population cultivated inthe absence of biomaterials. It appears, therefore, that PHO- b -DEG stimulated DNA replication and cell division.While the influence of PHAs on cell cycling has notpreviously been reported, Cheng et al. have reported changesin cell cycling due to the presence of 3-HBA. 10 They foundthat low concentrations of 3-HBA stimulated cell proliferationwith a concentration-dependent increase in the percentage of cells in the S phase. Thus, the monomeric component of PHB, Figure 5.  Summary of relative cell growth populations determinedfrom data in Figure 2 expressed as percentages of total populations(asterisk indicates statistically significant differences from asynchro-nous growth in serum). BioPEGylation of Polyhydroxyalkanoates  Biomacromolecules, Vol. 9, No. 10, 2008   2723
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