Influence on Progression of Stem Cell Cycle - American Chemical

Biomacromolecules 2010, 11, 2707–2715

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Polyhydroxybutyrate and its Copolymer with Polyhydroxyvalerate as Biomaterials: Influence on Progression of Stem Cell Cycle Tania Ahmed, Helder Marçal, Melissa Lawless, Nico S. Wanandy, Alex Chiu, and L. John R. Foster* Bio/Polymers Research Group, Centre for Advanced Macromolecular Design, School of Biotechnology and Biomolecular Sciences University of New South Wales, Sydney, Australia Received July 7, 2010; Revised Manuscript Received September 1, 2010

Poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) are biopolyesters reported to provide favorable microenvironments for cell culture and possess potential for tissue engineering applications. Both biopolymers have been investigated for applications in a variety of medical scenarios, including nerve and bone repair. This study investigated the influence these biomaterials exerted on cell cycle progression of olfactory ensheathing cells (OECs) and mesenchymal stem cells (MSCs) commonly used in the engineering of nerve and bone tissues. Cell cycle regulation is important for cell survival; analysis revealed that the biomaterials induced significant cell cycle progression in both MSCs and OECs. Significantly higher percentages of cells were cycled at synthesis (S) phase of the cycle on PHBV films compared to PHB, with MSCs more susceptible than OECs. Furthermore, detection of early stages of apoptotic activation showed significant differences in the two cell populations exhibiting necrosis and apoptosis when cultivated on the biomaterials. OECs compromised on PHB (5.6%) and PHBV (2.5%) compared to MSCs with 12.6% on PHB and 17% on PHBV. Significant differences in crystallinity and surface rugosity were determined between films of the two biomaterials, 88% and 1.12 µm for PHB and 76% and 0.72 µm for PHBV. While changes in surface properties may have influenced cell adhesion, the work presented here suggests that application of these biomaterials in tissue engineering are specific to cell type and requires a detailed investigation at the cell-material interface.

Introduction Biomaterials can serve as scaffolds that provide an ideal platform for tissue engineering applications. These scaffolds act as an extracellular matrix to support cellular growth and attachment by mimicking the in vivo environment.1 The primary aim of using such scaffolds in tissue engineering is to repair or reconstruct tissues and organs.2 As a result, these biomaterial devices can provide mechanical support as well as control cellular behavior by promoting cell adhesion, proliferation, and organization for the formation of functional tissue.3,4 A number of biopolymers from the family of polyhydroxyalkanoates (PHAs) have been investigated as potential candidates for such roles.5 Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by a wide variety of microorganisms under unbalanced growth conditions. Some PHAs have been shown to be biodegradable and biocompatible, supporting cell growth as well as guiding and organizing the cells, making them attractive as scaffolds for tissue engineering. PHAs have been investigated for various medical devices such as sutures, suture fasteners, repair patches, and bone plates.6 Frequently studied PHAs include poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), which are reported to provide favorable microenvironments for cell culture.4 Both PHB and PHBV have been investigated for potential applications in nerve and bone repair.7-13 We have recently shown that bioPEGylation of polyhydroxyoctanoate (PHO) not only permitted manipulation of the * To whom correspondence should be addressed. Tel.: +61(2)-93852054. Fax: +61(2)-9385-1483. E-mail: [email protected].

physiochemical and material properties of the resulting naturalsynthetic hybrid copolymer (PHO-b-PEG) but also influenced cellular responses.5 These results reinforce the need to gain an understanding into the mechanisms employed by cells to respond and adapt to different microenvironments. Despite the wealth of research investigating the design and development of polyhydroxyalkanoate-based biomaterials and their devices for biomedical applications, studies examining the influence of the biopolymers themselves on stem cells and their cycle and apoptosis are limited; these biological characteristics are important mechanisms in considering biocompatibility and tissue engineering potential of these biomaterials. Similarly, while there are a number of studies reporting the cultivation of various cell lineages, such as Macrophage,14 MC3T3 osteoblastic cells,7 Schwann cells,11 and MSCs15-17 on PHB and P(HB-co-8HV), none of these have applied a cell cycle approach. In the work reported here, we compare the physiochemical and material properties of PHB and P(HB-co-8HV) and their influence as scaffolds for olfactory ensheathing cell (OEC) and mesenchymal stem cell (MSC) lineages. This preliminary test for biocompatibility was determined by investigating the early stages of apoptotic activation and examining the influence of the biopolymers on cell cycle progression. While PHB-based scaffolds have been investigated as nerve conduits for OECs and MSCs, to the best of our knowledge, this is the first time that the influence of the biomaterial on cell cycle progression has been investigated on PHB and PHBV films. Cell cycle and apoptosis are both prerequisites for monitoring cellular growth and proliferation. As cells spread and populate the scaffold through cell proliferation, they go through various phases of cell cycle: rest (G0/G1), DNA replication (S), DNA repair (G2),

10.1021/bm1007579  2010 American Chemical Society Published on Web 09/17/2010

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and finally, cell division (mitosis, M). These cellular processes determine the behavior and fate of cells. In the olfactory epithelium, neural progenitor cells, OECs selfrenew themselves, proliferate, migrate, and differentiate through the process of neurogenesis, which is influenced by external factors.18 Environmental signals govern the survival and differentiation of neural progenitor cells by causing the cells to re-enter the cell cycle and influence cells in the immediate surroundings to differentiate into neurons.19 OECs are currently recognized as the gold standard in tissue engineering applications due to their capability to promote axonal regeneration and functional recovery after injury to spinal cord.20,21 Following injury and during nerve regeneration, the OECs leave the quiescent stage of the cell cycle and re-enter the cycle to proliferate to regenerate tissue. Eventually, submucosal-derived OECs reach a stage where they repopulate to form neurons of the olfactory epithelium.22 Similarly, MSCs isolated from bone marrow self-renew, proliferate, migrate, and differentiate into different tissue types of mesenchymal origin (osteocytes, chondrocytes, and adipocytes), and there is increasing recognition in their potential for neuronal repair.23 Thus, the influence of biomaterials such as PHB and P(HBco-8HV) on the progression of the cell cycle for such stem cells is of substantial interest in biocompatibility studies to assist in gaining broader understanding of the potential of OECs and MSCs in various applications of regenerative medicine.24,25

Materials and Methods Materials and Reagents. Mammalian cell growth medium, fetal bovine serum (FBS), and penicillin/streptococcus antibiotic were obtained from Gibco-Invitrogen (Sydney, Australia). OECs were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM/ F12) supplemented with 10% FBS purchased from Lonza (U.S.A.). Trypsin was acquired 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 prepared in dimethyl sulfoxide (DMSO) and stored at -20 °C. Naturally produced PHB and PHBV were obtained from Sigma-Aldrich, (Sydney, Australia) while chemicals including acidic acid and chloroform were of analytical grade and acquired from Univar (Sydney, Australia). Olfactory Ensheathing and Mesenchymal Stem Cell Cultures. Cells were cultivated according to protocols established by Marçal et al.5 Briefly, cells were cultured in sterile T75 tissue flasks containing DMEM-10% FBS and harvested at approximately 90% confluence. The spent media was aspirated and rinsed twice with 10 mL of phosphate-buffered saline (PBS). Trypsin (2 mL, 2.5%) was added to detach the cells and incubated at 37 °C with 5% CO2 for 2 min. Fresh, sterile medium (10 mL) was added to deactivate the trypsin and the contents were subsequently transferred to sterile 15 mL centrifuge tubes before centrifuging (10 min, 300 g). Supernatants were decanted and cell pellets were suspended in 10 mL of fresh, sterile culture medium. The cell volume was divided by transferring 5 mL aliquots to new T75 tissue culture flasks each containing 10 mL of sterile medium. Cells were immediately incubated at 37 °C with 5% CO2. Film Fabrication. PHB and PHBV films were prepared by dissolving 1 g of polymer in 80 mL of chloroform in clean Schott bottles which were subsequently sealed before heating with constant stirring (65 °C, 200 rpm, 3 h). The solutions were then allowed to cool by standing for 10 min at room temperature before being poured into clean, glass tissue culture dishes (100 mm diameter). The dishes were loosely covered with aluminum foil and left to stand under clean conditions for 72 h at room temperature (22 °C). Films which were flat, well distributed and maintained their integrity were selected for cell culturing.

Ahmed et al. Prior to cell culturing, films were gamma irradiated for 20 min at a dosage rate of 0.564 kGy/h. Film Characterization: Material Properties. An Instron 2752-005 tensiometer (Norwood, PA, U.S.A.) was used to measure the material properties of the solvent cast films. Polymer film strips (40 × 15 mm) were held between two pneumatic clamps positioned at a distance of 2.5 cm and pulled apart at a constant cross head speed of 20 mm/min until break. The force and elongation was measured and calculated using Bluehills software (version 2.0). Means of 12 test replicates for each film were calculated (n ) 12). Film Characterization: Surface Morphology. The surface of the polymer films were visualized using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Cells grown on PHB and P(HB-co-8HV) films were also visualized using SEM. Cells cultivated on the polymer films over 24 h had their aspirated medium removed and the specimens subsequently rinsed with PBS twice prior to fixing in PBS containing 2.5% glutaraldehyde solution (pH 7.4) for 4 h. The specimens were then rinsed three times in PBS for 5 min each before they were dehydrated stepwise in increasing concentrations of ethanol (from 30, 50, 70, 90, and 95 to 100%) for 10 min at each concentration. Films in 100% ethanol were gently cut into 11 × 11 mm squares before critical point drying then mounting on aluminum stubs and sputter coated with gold (2 min, 20 mA). Specimens were examined using a scanning electron microscope (Hitachi S3100N, Japan) at 15 kV and 750 mA. This protocol is modified from Wang et al.26 The microtopographies of PHB and P(HB-co-8HV) films were mapped using the reflection mode of a confocal scanning laser microscope (CSLM, Leica model TCS-SP, Germany) at excitation and emission wavelengths of 458 and 440-470 nm, respectively. Multiple images through the z-plane were recorded (step size ) 2.5 µm). A total of 30 images were taken to generate a 3D depth map and calculate the average surface roughness values (Ra). The Ra values for the films were calculated from the images using ImageJ software (National Institute of Health, U.S.A.) according to ISO 4298 (2000):

Ra ) 1/L

∫

L

0

|z|dx

(1)

where the average surface roughness (Ra) was calculated from the sampling length (L), and the plane (z) and the variations of irregularities (dx) from the mean line.27 Cell Cycle and Apoptosis. Cells were cultured in DMEM medium containing 10% FBS in glass tissue culture dishes coated with films of PHB and P(HB-co-8HV) and grown for 5 days with a working volume of 11 mL. Controls in the absence of the polymers were simultaneously conducted from the same inoculums. Additional controls were similarly performed to validate the acquired data. Controls for DNA content were as follows: (1) cells in 10% FBS, (2) serum-deprived cells, (3) cells synchronized for 24 h in medium containing 10% FBS and 1 µg/mL aphidicolin, and (4) 2 µM nocodazole. During harvesting, cells that were subjected to a DNA content assay were centrifuged at 300 g and washed once in PBS before being resuspended with cell cycle staining buffer at a concentration of 1 × 106 cells/mL and incubated on ice for 30 min before acquisition using flow cytometry. Control for apoptosis detection was also conducted by employing cells that were subjected to 2 µM nocodazole for 24 h in the presence of 10% FBS, while controls of healthy cells were also conducted using the same conditions with the absence of apoptosis inducing agents. Cell numbers and their viability were determined using the trypan blue exclusion method, as per Marçal et al.5 Apoptotic indices associated with the externalization of phosphotidylserine (PS) were assayed with an FITC-conjugates Annexin-V apoptosis kit and counterstained with propidium iodide (PI; BD Bioscience, Pharmingen, U.S.A.). Staining was conducted in accordance with the manufacturer’s protocol. Prior to analysis, cells were harvested and washed once in PBS. FITC and PI molecules were visualized using

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Figure 2. Graph of cell numbers after cultivation in the presence of PHB and P(HB-co-8HV); initial seeding of 500000 cells: (gray) viable cells, (black) nonviable cells.

Figure 1. SEMs of Olfactory ensheathing cells (a-c) and Mesenchymal stem cells (d-f) cultivated in the absence of biomaterials; asynchronous growth (a) and (d), and on PHB (b,e) and P(HB-co8HV) films (c,f); Arrow illustrates filipodia.

a Becton and Dickinson FACS Calibur Flow Cytometer, equipped with a 488 nm laser. Bivariate forward and side-angle scatter gating were used to identify homogeneous populations while excluding cellular debris and dead cells. FITC and PI fluorescence’s were visualized using 530/30- and 585/42-bandpass filters, respectively (10000 events for Annexin-V and 50000 events for a DNA content histogram was collected). Photomultiplier (PMT) voltages were adjusted to ensure that autofluorescence associated with nonapoptotic samples described a Gaussian distribution within the first two log-decade on univariate histograms. Analysis was performed using Cytomation Summit v3.1 software (Cytomation, Fort Collins, CO). The PI staining solution was prepared by mixing 0.1% (v/v) Triton X-100, 0.1% (w/v) BSA, 40 µg mL-1 PI, and 10 µg mL-1 RNase A in PBS. Cells were extracted through centrifugation at 300 g, rinsed once in PBS, and centrifuged again. Cells were then resuspended, transferred to FACS tubes, and incubated in the staining solution for 30 min on ice. PI fluorescence intensities were deconvulated using ModFit (Verity Software House, Inc., Topsham, ME) to resolve cell cycle distributions. All cell studies were run in triplicate and repeated (n ) 3 × 2). Statistical Analysis. All data was statistically evaluated using the two-way ANOVA analysis and Bonferroni post-test (significance level: 0.05).

Results and Discussion Cell Cycling. Cultivation of the OECs and MSCs on the PHA films revealed differences in cell proliferation and adhesion with respect to their cell types while retaining their normal morphology, as observed in standard culturing conditions (Figure 1a,d). Here, MSCs displayed a homogeneous triangular morphology in comparison to OECs, which exhibited a “mesh-like” neuronal structure due to their close proximity to one another. OECs grown on PHB and P(HB-co-8HV) films displayed a spindlelike morphology and were well spread across both films with

no noticeable differences (Figure 1b,c). However, cell counting showed that there were a greater number of cells cultivated on the P(HB-co-8HV) films compared to those on the PHB, suggesting that the adhesion and colonisation of these cell lines was influenced by the substrate (Figures 1e,f and 2). Actin-containing filopodial extensions, considered to be structurally important for cellular motility and guidance, were apparent on the P(HB-co-8HV) films for both OEC and MSC lineages (Figure 1c,f).28,29 These filopodia allow cells to move directionally, establishing greater cell to cell communication.30 Furthermore, neurite sprouting of the OECs on the P(HB-co8HV) film was also observed by the presence of extensive pseudopods (Figure 1c). These observations suggest that both cell lineages preferred films of P(HB-co-8HV) to those of PHB and this was most apparent with the MSCs; this qualitative assessment was supported by quantitative analysis of cell counts (Figure 2) and their cell cycles. While a number of studies have investigated PHA biocompatibility in vitro using various cell lineages, there are no reports on the influence of these biomaterials on the progression of the cell cycle.6,31 However, given the importance of cell cycle regulation for cell survival, we have investigated the influence exerted by PHB and P(HB-co-8HV) on the cycles of OECs and MSCs. Regulation of the cell cycle involves processes crucial to cell survival, including the detection and repair of genetic damage as well as the prevention of uncontrolled cell division, that is, tumorgenesis. In tissue engineering, the temporal and spatial control of cell cycles for cell growth, differentiation, and migration is considered a major challenge. The influence of biomaterials on such stem cell cycling is, therefore, of considerable interest as these materials are considered for tissue engineering where the cells spread and populate through the biomaterial scaffold, cycling through the various phases of cell cycle: quiescence (G0/G1), DNA replication (S), rest (G2), and finally, cell division (mitosis, M). In our study, OECs and MSCs cultivated on films of PHB and P(HB-co-8HV) displayed typical DNA content profiles illustrated by a distribution that mainly consisted of cells in G0/ G1 and S phases. However, the DNA content of the OECs and MSCs demonstrated significant cell cycle aberrations between the two cell types (Figure 3). A qualitative analysis of the FACS histograms in Figure 3 clearly shows that the cells cycled

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Figure 3. FACS histograms of olfactory ensheathing cell populations with DNA exhibiting G0/G1, G2-M, and S growth phases. Positive controls for G0/G1 phase through serum deprivation (a), S phase through addition of aphidicolin (b), G2/M phase through addition of Nocodazole (c), asynchronous growth (d), and for cells cultivated on films of PHB (e) and P(HB-co-8HV) (f).

through G0/G1, S and G2-M phases. In Figure 3d, OECs displaying asynchronous growth gave a typical DNA content profile illustrated by a distribution that mainly consisted of cells in G0/G1 (72.4%) and S-phase (27.6%). Similar results were obtained for asynchronous MSCs (Figure 4). In contrast, positive controls had their cell cycle distribution altered and were consistent with expected DNA content profiles following treatments with the corresponding arresting agents. Conditions of serum deprivation maintained the cells in G0/G1 quiescent phase (92.7%, Figures 3a and 4a) while the addition of synchronizing agents, aphidicolin, and nocodazole arrested OECs at the G1/S (79.1%, Figures 3b and 4b) and G2/M (32.1%, Figures 3c and 4c) phases, respectively. In comparison to these controls, OECs cultivated on the PHB and P(HB-co8HV) films showed no significant inhibitions in their growth (Figure 3e,f). However, the distribution profiles of DNA content for MSCs cultivated on the PHA films revealed significant differences in their populations with the presence of apoptotic cells when cultivated on PHB (Figure 4e,f). Figure 5 shows a quantitative analysis of the histogram data, for both OEC and MSC cultivations the positive controls

revealed significant differences (p < 0.05) when compared to the control without biomaterials, (i.e., asynchronous growth). OECs cultivated on the PHA films showed no significant variations in the distribution of cell populations when compared to those cultivated under asynchronous conditions, with populations in the quiescent and S phases of the cell cycle (Figure 5a). In contrast, growth of MSCs on the PHA films varied significantly from their asynchronous growth control with a greater proportion (26.7%) of cells arresting in the repair phase (p < 0.05, Figure 5b). It has been previously reported that the monomeric component of PHB, 3-HBA have an influence on cell proliferation32 and this in turn may have affected cell cycling.33 However, while examination of the film surfaces using electron microscopy showed significant differences in surface morphology, no apparent degradation could be discerned (Figure 5). Thus, PHB surfaces had an apparently inhibitory effect on cell growth and proliferation, particularly the MSCs which is supported by the scanning electron micrographs and cell counts as discussed earlier. The influence of PHB surfaces on cell attachment have been reported by Foster et al. who cultivated



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Figure 4. FACS histograms of mesenchymal srem cell populations with DNA exhibiting G0/G1, G2-M and S phase growth phases. Positive controls for G0/G1 phase through serum deprivation (a), S phase through addition of aphidicolin (b), G2/M phase through addition of Nocodazole (c), asynchronous growth (d), and for cells cultivated on films of PHB (e) and P(HB-co-8HV) (f).

human epithelial cells on films of untreated and treated centrifugally spun PHB fibers.34 Surface treatment of PHB fibers with dilute acidic or alkali solutions, (3% sulphuric acid and sodium hydroxide, respectively) promoted cellular adhesion and proliferation on PHB due to changes in surface charge. Furthermore, the comparative reduced cellular growth on untreated PHB fibers suggests a reduction in cell viability and increased apoptosis, consistent with the data found here.34,35 Apoptotic Indices. Annexin V assay was used to detect the early stage of apoptosis, which is important for understanding programmed cell death.36 Annexin V is a calcium-dependent phospholipid binding protein that preferentially binds to phospholipid phosphatidylserine (PS). Most mammalian cells have PS within the plasma membrane, which is externalized once apoptosis is initiated and subsequently detected on the cell surface by staining with an Annexin V FITC (fluorescein isothiocyanate) conjugate of high affinity.37 Cells were categorized according to their apoptotic/necrotic fluorescence profiles as nonapoptotic, membrane compromised, apoptotic, and necrotic. Both cell lineages showed some degree of membrane

compromise when cultivated on the two PHA types, with this condition more noticeable with the MSCs (Figure 6). Cells cultured in serum did not show significant statistical differences between the two cell populations and were relatively consistent with expected results, with the majority of cells remaining intact, viable, and nonapoptotic (Figure 7). However, cells cultivated with nocodazole exhibited significant differences between the OECs and MSCs (p < 0.05). Nocodazole-induced OECs to undergo both early and late apoptosis, represented by increases in the Annexin-V FITC and PI positive populations of 4.8 and 4.2% respectively. In contrast, there was a marked decrease in early apoptosis (0.8%) and a significant increase in late apoptosis (7.5%, Figure 7a). Significant deviations were also observed between OECs and MSCs exhibiting apoptosis and necrosis when cultivated in the presence of the biomaterials. Lower percentages of MSCs were early apoptotic on PHB (1.6%) and PHBV (0.6%) in comparison to OECs, which displayed a similar apoptotic profile as the positive control (nocadazole). However, the majority of MSCs had their plasma membrane compromised indicated by an influx of PI across the

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Figure 5. Percentage distribution of DNA content in G0-G1 (gray with black), G2-M (gray with light gray) and S (gray with dark gray) growth phases for olfactory ensheathing cells (a) and mesenchymal stem cells (b). Summary of DNA content relative to the cell growth populations determined from data in Figures 3 and 4 expressed as percentages of total populations (*indicates significant differences from asynchronous growth in serum, p < 0.05).

Figure 6. Fluorescence profiles of olfactory ensheathing cell (a,b) and mesenchymal stem cell (c,d) populations gated to exhibit different stages of apoptosis and necrosis when cultivated of PHB (a,c) and P(HB-co-8HV) (b,d).

permeable membrane, with a higher percentage on PHBV (17.0%) than on PHB film (12.6%, Figure 7b). This result was

Ahmed et al.

Figure 7. Percentages of relative populations of OECs (a) and MSCs (b) that are apoptotic (gray with dark gray), nonapoptotic (gray with very light gray), necrotic (gray with light gray), and membrane compromised (gray with black) when cultivated in control media and in the presence of PHB and P(HB-co-8HV) films (*significant differences from asynchronous growth in serum, P < 0.05).

reversed for the case of OECs with a greater proportion of cells being compromised on PHB (5.6%) than PHBV film (2.51%, Figure 6a). Our data suggests that both PHB and PHBV elicit the OECs and MSCs to undergo early stages of apoptotic activation, however, a greater percentage of OECs exhibited early apoptotic activation than MSCs on PHB and PHBV films. Polymer Properties. The nature of biomaterial surfaces has been shown to be critical for cell attachment.38 Nucleation of PHB and PHBV films is known to influence the surface, physiochemical, and material properties of films fabricated from these biomacromolecules; these are important design criteria for biomaterial devices.33 For example, material strength is a crucial component for the fabrication of suitable scaffolds for use in nerve and cartilage repair.39 Thus, biomaterials should be capable of withstanding the physical and mechanical pressures associated with the device function as well as supporting cellular growth and proliferation without disintegrating in the surrounding medium. In the study reported here, tensile testing was conducted to determine the material strength of the PHB and PHBV films.40 Consistent with previous studies, the P(HBco-8HV) films were significantly more flexible than their PHB counterparts, displaying a tensile strength of 24.15 ((6.75) compared to 18.63 ((4.07) MPa (p < 0.05, Table 1).39 Differences may be due to variations in HV contents and fabrication technique. Wang et al. examined PHB capsules rather than films and a higher HV content (30 wt %) of PHB/PHBV capsules. Furthermore, PHBV exhibited a greater extension to break than PHB, 5.15% ((1.51) and 3.31% ((1.98) respectively. These differences in material properties are in part due to crystallization behavior. Consistent with reported studies, the



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Table 1. Summary of Material and Surface Properties for Solvent Cast Films of PHB and P(HB-co-8HV)a parameter

PHB

P(HB-co-8HV)

tensile strength (MPa) extension to break (%) crystallinity (%) average surface roughness (µm) microroughness waviness contact angle (deg)

18.63 ((4.07) 3.31 ((1.98) 88 ((4) 1.14 ((0.18) 0.64 ((0.07) 0.34 ((0.05) 73.3 ((0.52)

24.15 ((6.75) 5.15 ((1.51) 76 ((3)* 0.72 ((0.15)* 0.70 ((0.08) 0.32 ((0.04) 75.3 ((0.68)

a

*Significant differences between the two biomaterials, p < 0.05.

PHA films in this study were crystalline, 88 and 76% for PHB and PHBV, respectively (Table 1).26 There is growing recognition that surface architecture is one of the predominant determinants in influencing cell colonization and adhesion.41 Surface morphology modulates the extent of cell spread which is a major determinant of cell growth and function, thus subtle changes in topological features can translate into significant differences in cell behavior.7,42,43 Maintenance of cellular behavior requires the regulation of the highly organized cell cycle responsible for controlling cell fate. However, only a few studies present analytical data quantitatively measuring surface morphology. We have previously reported the application of confocal laser scanning microscopy (CLSM) to analyze surface rugosities of biopolymer based films in the acknowledged standard of “average surface roughness” (Ra).5 Average surface roughness is defined as “the average departure of the surface from the mean surface plane at a given moment” (ISO4287:1997). In the study here, surface morphology was qualitatively and quantitatively investigated using a combination of SEM and CLSM. Figure 8 shows electron micrographs of the PHA films; the PHB film surface appears comparatively more irregular than that of P(HB-co-8HV). The qualitative SEM observations were supported by 3D mapping of the polymer film surfaces with CLSM (Figure 9). Analysis of the microtopographies shown in Figure 8 gave Ra values of 1.14 ((0.18) and 0.72 ((0.15) µm for the PHB and P(HB-co-8HV) films, respectively. The surface rugosity of the PHA films was further analyzed for structures under 5 µm defined as “microroughness” and those larger than 5 µm exhibiting a degree of harmonics termed “waviness” (ISO4287:1997, Table 1). There was no significant difference in these components between the two biomaterials. Eginton et al. reported that hydrophobicity rather than surface roughness influenced biofilm attachment to polyethylene films.44-46 In the experiments reported here, mean contact angles of the PHA films were determined as a measure of surface hydrophobicity, with little difference between PHB (73.3°) and P(HB-co-8HV) (75.3°) observed. However, the slightly higher surface hydrophobicity for the P(HB-co-8HV) films used in these experiments is consistent with Choi et al. who reported increases in surface hydrophobicity with valerate content.47

Figure 8. SEMs of PHB (a) and P(HB-co-8HV) (b) film surfaces illustrating surface morphologies.

Conclusions The convergence of biomaterial engineering and stem cell research in the field of tissue engineering holds great promises to revolutionize regenerative medicine. Implementation of tailored biomaterials to support the growth of appropriate cells requires these scaffolds to be biocompatible, biodegradable, and immunologically inert. PHAs have been extensively studied as biomaterials possessing a diverse range of properties, allowing

Figure 9. 3D Microtopographies of PHB (a) and P(HB-co-8HV) (b) film surfaces, as determined through confocal laser scanning microscopy illustrating surface topographies.

them to be produced with favorable properties; in contrast, assessment of their biocompatibility in terms of cell cycle and apoptosis has yet to be reported.

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In the study reported here, qualitative examination of the cellular morphology of OECs and MSCs on films of PHB and P(HB-co-8HV) using SEMs and cell counting data suggested a positive influence exerted by P(HB-co-8HV) on cellular growth and adhesion, with cells maintaining their morphology, as observed in standard culturing conditions. In contrast, a reduction in cellular adhesion and migration was observed on PHB films. Analysis of cell cycle confirmed that these PHA biomaterials are biocompatible, with OECs and MSCs displaying no cellular aberrations. However, detection of early stages of apoptotic activation revealed that both PHB and P(HB-co-8HV) elicited the cells to undergo early apoptosis and necrosis. With further clarification of the cellular mechanisms adopted by both OECs and MSCs, a comprehensive understanding will be provided on the biocompatibility and potential of these PHAs for the repair and engineering of nerve and bone tissues.

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Influence on Progression of Stem Cell Cycle - American Chemical

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