Sterilization Techniques Influence Physical Properties and Cell-Biomaterial Interactions in Plasticized Polylactide Foam Scaffolds

Transcription

1 Sterilization Techniques Influence Physical Properties and Cell-Biomaterial Interactions in Plasticized Polylactide Foam Scaffolds Matthieu Cuenoud, PhD 1, Salim Elias Darwiche, PhD 1, John Christopher Plummer, PhD 1, Lee Ann Applegate, PhD 2, Jan-Anders Manson, PhD 1, Pierre-Etienne Bourban, PhD 1, Dominique Pioletti, PhD 1. 1 Ecole Polytechnique Federale de Lausanne, Lausanne, Switzerland, 2 Lausanne University Hospital, Lausanne, Switzerland. Disclosures: M. Cuenoud: None. S.E. Darwiche: None. J. Plummer: None. L. Applegate: None. J. Manson: None. P. Bourban: None. D. Pioletti: None. Introduction: The effect of sterilization methods on cell-biomaterial interactions is an often overlooked but fundamental aspect in translating a biomaterial from bench to clinic. Due to their sensitivity to high temperature, pressure, and moisture, degradable polyesters require special consideration during sterilization and stocking to best preserve the properties of the biomaterial. Gamma ray irradiation and ethylene oxide gas treatment have emerged as popular sterilization tools with important tradeoffs nevertheless. The former has been shown to cause degradation of polymer chains with a reduction in molar mass and therefore a loss of mechanical properties and the latter treatment, milder than ionizing radiation, may potentially leave harmful toxic residues. The effect of either sterilization method on cell adhesion, proliferation and interaction with bioactive materials is still poorly understood and may be crucial to a successful translation of a biomaterial to the clinic. The object of the present study is to assess the impact of different sterilization methods on the in vitro biological response to foams produced from PLLA and PLLA plasticized with PEG. This not only represents a crucial step in the development of new materials aimed specifically at osteochondral regeneration, but also is intended to provide broader insight into the consequences of the various sterilization methods on biomaterial processing and biocompatibility. Methods: Poly-L-Lactide foams and plasticized blends containing 20 wt% Polyethylene Glycol (PLLA20PEG) foams were sterilized either by exposing samples to kgy of gamma rays (Leoni Studer Hard AG, Switzerland) or by treatment with ethylene oxide gas following a standard regimen of preconditioning, gasing and aeration while maintaining the temperature at 55 C throughout the 4 hour treatment (University Hospital of Lausanne, Switzerland). Scaffolds were then placed in basal growth medium containing Dulbecco's Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (Sigma, St. Louis, MO) for 1.5 hours at 37 C to allow hydration followed by 20 mins in a water basin sonicator. We have previously reported the reliable expansion and characterization of a clinical-grade human epiphyseal chondro-progenitor (ECP) cell bank from a single tissue donation [1]. As osteochondral progenitors, ECPs exhibit remarkable homogeneity and stability in expansion, which motivated their use in this investigation. As such, ECPs were seeded onto the foam scaffolds to assess for biocompatibility, viability, proliferation, adhesion and spreading. Samples were harvested after 48 hours to assess cell adhesion and seeding efficiency. Cell-seeded scaffolds were fixed in 4% paraformaldehyde and stained with AlexaFluor 488 conjugated Phalloidin to visualize actin fibers. Additional samples were fixed in a 2.5% solution of glutaraldehyde and processed for scanning electron microscopy to visualize both cell-biomaterial interactions and biomaterial topographical changes. Some samples were also kept in culture three and seven days after cell seeding and processed to check for cell viability and proliferation using the Cell Titer 96 Aqueous One Solution Cell Proliferation Reagent (Promega, Fitchburg, WI, USA). Results: No long-term toxicity was detected from the scaffold materials. The amount of cells present at any given time was markedly lower in ethylene oxide sterilized scaffolds versus gamma sterilized scaffolds. Gamma sterilization may have affected surface roughness, which influenced cell-biomaterial interaction. Across formulations, the most apparent differences appeared in gamma-sterilized scaffolds, where PLLA foams showed a stark advantage over PLLA20PEG. PEG polymers have indeed been known to act as inert biomaterials and might inhibit cell adhesion. PEG containing blends might have also responded differently to ionizing radiation or ethylene oxide gas exposure compared to pure PLLA. Mirroring the trend seen with cellular metabolic levels 3 days after seeding, cells exhibited a stretched morphology with prominently developed stress fibers and anchor points to the underlying matrix in gamma sterilized scaffolds (Figure 1). This behavior was also seen to a lesser extent on gas sterilized PLLA scaffolds. In contrast, cells attached on gas sterilized PLLA20PEG scaffolds 48 hours after seeding were notably sparse and preferentially exhibited rounder morphologies and reduced spreading. Cellular projections, indicative of active cell-cell communication and an increased potential for migration, proliferation and substrate colonization, were also lacking in these two groups. Comparing cellular morphologies across polymer compositions, PLLA scaffolds allowed for extensive cellular anchoring and harbored cells exhibiting long projections, spread morphologies and prominent stress fibers. Visualizing adherent cells using scanning electron microscopy revealed particular patterns of cell spreading and attachment (Figure 2). On ethylene oxide sterilized scaffolds, cells were able to bridge gaps between pore walls and create cell projections, which did not seem to be restricted by the surface topography of the material. On the PEG containing counterpart of the ethylene oxide sterilized scaffold, cells were still able to spread, albeit preferentially in cell clumps. Adherent cells appeared able to bridge the chains of droplets

2 structures between PLLA spherulites. Gamma sterilized scaffolds exhibited more extensive cell spreading, more cell projections and, unlike ethylene oxide sterilized samples, cells showing a higher level of attachment to the material, despite areas of higher cell density. This pattern suggests that while spherulite geometry and droplet structures might not solely drive preferential cell attachment, cell-material interactions are very much affected by material composition and sterilization, putting the microtopography of porous foams at the forefront of cell seeding efficiency and perhaps even subsequent biological responses which may very well dictate the type of engineered biological tissue. Discussion: Sterilization methods used on PLLA and PLLA/PEG degradable polyesters heavily impacted cell-biomaterial interactions. Indeed, changes in cell adhesion, proliferation and interaction after sterilization were observed, allowing the use of sterilization techniques to modulate the physical properties of such substrates. The enhanced cell-substrate interaction observed in PLLA scaffolds, the architecture of which usually required dynamic cellular seeding before in vivo implantation might in fact play a fundamental role in optimizing their homogenous colonization by implanted cells. This would overcome the need for complex cell seeding protocols. Significance: Sterilization methods used on degradable polyesters heavily impact cell-biomaterial interactions. Studying these effects allows us to control the changes in cell adhesion, proliferation and interaction after sterilization, using these physical properties to differentially drive cell maturation in an osteochondral implant site and insure that what is implanted in vivo is compatible with what has been tested on the bench. Acknowledgments: These studies were funded by the Swiss National Science Foundation and the Interinstitutional Center for Translational Biomechanics EPFL-CHUV-DAL. References: [1] Darwiche SE, Scaletta C, Raffoul W, Pioletti DP, Applegate LA. Epiphyseal Chondro-Progenitors Provide a Stable Cell Source for Cartilage Cell Therapy. Cell Medicine. 2012;4:23-32.

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4 ORS 2014 Annual Meeting Poster No: 0269

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