Biomacromolecules 2008, 9, 2719–2726
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BioPEGylation of Polyhydroxyalkanoates: Influence on Properties 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 in hybrids with amphiphillic properties. However, the study of such natural-synthetic hybrids of PHA-b-PEG is still in its infancy. In this study, we report the influence of bioPEGylation of polyhydroxyoctanoate (PHO) on its physiochemical, material, and biological properties. Consistent with previous studies, bioPEGylation with diethylene glycol (DEG) showed a significant reduction in PHA molecular weight (57%). In comparison to solvent cast films of PHO, PHO-b-DEG films possessed a noticeable X-ray diffraction peak at 9.82° and increased Young’s modulus of 11 Gpa (83%). Potential biocompatibility was investigated by measuring the early phase of apoptosis in myoblastic satellite-stem cells (C2C12). Comparative analysis of cell proliferation and progression in the presence of the mcl-PHA and its hybrid showed that the latter induced significant cell cycle progression: the first time a biomaterial has been shown to do so. Microtopographies of the film surfaces demonstrated that these differences were not due to changes in surface morphology; both polymers possessed average surface rugosities of 1.4 ( 0.2 µm. However, a slight decrease in surface hydrophobicity (3.5 ( 0.9°) due to the hydrophilic DEG may have exerted an influence. The results support the further study of bioPEGylated PHAs as potential biomaterials in the field of tissue engineering.
Introduction Biomaterials used in tissue engineering applications serve as support scaffolds and adhesive substrates for cells during in vitro culture and subsequent to implantation.1 The design and selection of scaffolding biomaterials can significantly influence the development of engineered tissues. Various biopolymers have 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 microorganisms under unbalanced growth conditions and serve a carbon and energy storage function.5 To date, over 105 different monomeric structures have been identified as components of PHAs, but only several have been investigated for their potential as tissue engineering materials.6,7 Of these, the focus has been on PHAs comprised of monomers with relatively short chain lengths (4-6 carbon units, scl-PHA), mainly poly(hydroxybutyrate) (PHB), and its copolymers.7 The PHB homopolymer consists of hydroxybutyric acid (HBA) monomers with the microbial monomer chemically identical to mammalian HBA produced as one of the ketone bodies during prolonged starvation and under diabetic conditions.8 Recently, Chen and co-workers have speculated that 3-HBA supports tissue regeneration by preventing cell apoptosis.9,10 In contrast to scl-PHAs, studies on PHAs with monomers of medium chain lengths (6-16 carbon units, * To whom correspondence should be addressed. Tel.: (+61)2-93852054. Fax: (+61)2-9385-1483. E-mail:
[email protected]. † University of New South Wales. ‡ University of Queensland.
mcl-PHA) are limited and restricted to the terpolymer poly(hydroxyoctanoate) (PHO).7 PHAs and their composites have also been used to produce a variety of medical devices, from sutures and bone plates to vein valves, various wound dressings, and tissue regeneration devices (for a detailed review, see ref 7). In vitro and in vivo studies using PHA-based biomaterials have shown equivocal differences in biocompatibility and biodegradability.11,12 It has been suggested that differences in biocompatibility between PHAs may be due, in part, to variations in physical and morphological properties such as microtopography and surface hydrophobicity.13,14 Thus, a further understanding of the complex interface events that occur between cells and a biomaterial surface is necessary when engineering biopolymers. This is due to the influence that biopolymers induce on cellular responses at the material interface.15 Surface chemistry, in particular, is known to affect cell function and adhesion.16 It is therefore desirable to engineer polymers that are designed to incorporate modifications at the material surface that are suitable for their intended applications. Molecular mass and monomeric composition of PHAs and, by extension, their material properties, including surface chemistry, can be controlled through the addition of poly(ethylene glycol) (PEG).17 Furthermore, PEGs with molecular weights below approximately 600 can act as chain terminating agents resulting in the biosynthesis of natural-synthetic hybrids of PEGylated PHAs, that is, “bioPEGylation”.17 The majority of research in this field has focused on the PEG modulated control of PHA biosynthesis, little has been done to consider PHA-b-PEG hybrids as biomaterials.17 We have recently demonstrated that the hybrid polymer chains can be induced to
10.1021/bm800418e CCC: $40.75 2008 American Chemical Society Published on Web 08/29/2008
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Biomacromolecules, Vol. 9, No. 10, 2008
Marc¸al et al.
Table 1. Summary of Physiochemical, Material, and Biological Properties of PHO-b-DEG and its Solvent Cast Films Compared to PHO property
PHO
PHO-bDEG
molecular weight (×103) molecular weight number (×103) polydispersity index density (g/cm3) melting point (°C) glass transition temperature (°C) enthalpy of fusion (J/g) crystallinity (%) Young’s modulus (Gpa) tensile strength (Mpa) extension to break (%) water contact angle (°) Surface Tension (mN/m) surface tension (mN/m) γs1 average surface roughness (µm)
235 142 1.65 1.05 55 ((1.1) -32 ((1.4) 31 ((3.5) 38 ((3.4) 6 ((0.3) 7 ((0.7) 580 ((9.4) 73.8 ((0.9) 40 ((0.5) 17 ((0.4) 1.5 ((0.2)
100 77 1.30 0.99 55 ((0.8) -31 ((1.1) 34 ((2.7) 34 ((2.9) 11 ((0.4) 6 ((0.7) 540 ((8.3) 70.3 ((0.7) 42 ((0.4) 19 ((0.2) 1.3 ((0.2)
exhibit a degree of self-assembly for the fabrication of disordered microporous films that have potential for cell immobilization.17,18 In this study, we compare the physiochemical and material properties of PHO with its bioPEGylated hybrid “end-capped” with diethylene glycol (DEG as PEG106). Furthermore, we assessed the biocompatibility of PHO and PHO-b-DEG with myoblastic satellite-stem cells derived from skeletal muscle. This preliminary test for biocompatibility was determined by analyzing the early stages of apoptotic activation and investigating the influence of the polymers on cell cycle progression. To the best of our knowledge, this is the first time that the influence of the biomaterial on satellite-stem cell cycle has been investigated. The seeding of biopolymeric scaffolds with stem cells for the generation of tissue-engineered products requires two cellular processes to occur concurrently. Cells must spread and populate the scaffold through cell proliferation, whereby cells cycle through phases classified as “G0/G1” (resting), “S” (DNA synthesis), “M” (mitosis), and “G2” (gap between S and M). Typically, it is the G0/G1 phase that is shortened when cells move from a nonproliferative to a proliferative state, while S, G2 and M remain relatively constant. Concurrently, cells undergo differentiation whereby cells are programmed to fulfill specific functions. Skeletal muscle-derived satellite-stem cells are recognized to provide repair and regeneration following local injury of muscle fibers.19 In undamaged adult tissues, satellitestem cells are quiescent myocyte cells that are usually detected just beneath the basal lamina.20 However, following injury and during muscle regeneration, the satellite-stem cells leave quiescence and re-enter the cell cycle to proliferate as myoblastic cells to replenish the tissue. Ultimately, they reach terminal differentiation and fuse together to form myotubes and myofibers.21 Manipulation of their growth using biomaterials is therefore of considerable interest, as is the suitability of such biomaterials in tissue engineering.
Materials and Methods Materials. Mammalian cell growth medium and fetal bovine serum (FBS) were purchased from Gibco-Invitrogen (Sydney, Australia). Stem cells (C2C12) were routinely cultured in Dulbecco’s Modified Eagle’s Medium (DMEM/F12) supplemented with 10% FBS. Trypsin was purchased from Sigma-Aldrich (St. Louis, MO) and cell arresting agents aphidicolin and nocodazole were purchased from Sigma-Aldrich (Sydney, Australia). Stock solutions of aphidicolin and nocodazole were
Figure 1. Graph showing change in molar volume with increasing mole 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 as produced using P. oleovorans.
prepared in dimethyl sulfoxide (DMSO) and stored at -20 °C. A kit to determine endotoxin levels was purchased from Sigma (Sydney, Australia). Octanoic acid, DEG (as PEG106), and microbially produced PHB were purchased from Sigma-Aldrich (Sydney, Australia). All other chemicals 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 in sterile T25 tissue flasks were subcultured in DMEM-10% FBS medium where they attained approximately 90% confluence. The spent media was 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 for 2 min. Cells were subsequently dislodged and transferred to sterile centrifuge tubes before centrifuging (10 min at 300 × g). Supernatants were decanted, and cell pellets were resuspended in 10 mL of fresh medium. Of this, 1 mL aliquots were transferred to new T-flasks, each containing 9 mL of fresh medium (i.e., 1 in 10 dilutions). Cells were immediately incubated at 37 °C after gassing with 5% CO2 for approximately 10 s. Polymer Production. PHO and PHO-b-DEG were produced through simultaneous cultivations of Pseudomonas oleoVorans (ATCC 29347) using the same inoculum, as described by Foster et al.18 Octanoic acid was used as a carbon source and 2% (w/v) DEG was added for the production of the bioPEGylated hybrid. Polymer samples were extracted into chloroform from the lyophilised biomass and precipitated into cold methanol. The samples were subsequently purified through a series of chloroform solvation and methanol precipitation cycles.22 Purification was established by measuring the endotoxin content for each cycle.
BioPEGylation of Polyhydroxyalkanoates
Figure 3. Microscopic images of myoblastic satellite-stem cells cultivated on films of PHO-b-DEG (a) and PHO (b); bar ) 200 µm.
Polymer compositions and bioPEGylation were confirmed using gas chromatography (GC) and 2D nuclear magnetic resonance spectroscopy (1H-1H COSY and 1H-13C HSQC), as described in detail previously.18 Briefly, polymer samples were dissolved in deuterated chloroform (∼4 mg/mL) and then examined using a Bruker DMX600 (600.13 MHz for 1H and 150.92 MHz for 13C). 1H spectra were recorded with a pulse width of 4.5 ms (458 pulse), a spectral width of 6.6 kHz, an acquisition time of 2.5 s, and a relaxation delay of 6 s. Between 64 and 256 scans for the required signal-to-noise correction were collected and all scans were referenced internally to chloroform (7.26 ppm with respect to tetramethylsilane). Samples of purified PHO and PHO-b-DEG were dissolved in analytical-grade chloroform and fabricated into thin films by casting into clean, dry, sterile glass Petri dishes, dried for 48 h in a vacuum desiccator and removed from the dishes before allowing to stand for an additional 48 h until their weights had atmospherically equilibrated. The films were then aged for an additional three weeks to enable their crystallinity to reach equilibrium. For standardization purposes all microscopic imaging and cell cultivation was performed on film sides that were cast in contact to the clean glass. Polymer Characterization. The molecular mass properties of the polymers were determined by gel permeation chromatography using an LC-10ATVP Shimadzu solvent delivery system combined with a SIL-10ADVP Shimadzu autoinjector possessing a stepwise injection control and a column set consisting of a PL 5.0 mm bead size guard column 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 Column Oven and an RID-10A Shimadzu refractive index detector. Samples were analyzed in a continuous phase of tetrahydrofuran (THF, 1 mL/ min) and values were calculated from a calibration curve of polystyrene standards with low polydispersity (
