Osteoblast response to PLGA tissue engineering scaffolds with PEO modified surface chemistries and demonstration of patterned cell response
Introduction
A common paradigm in the field of tissue engineering is that new tissue can be generated by combining cells with a tissue engineering scaffold. The ultimate goal is for the cells to grow within, and usually replace, the scaffold to form a new tissue that is functionally equivalent to the desired tissue. Because tissues are characterized by a well-defined three-dimensional arrangement of cells, a tissue engineering scaffold that facilitates the organization and differentiation of the new tissue will have improved performance in comparison to a scaffold that only provides space and appropriate surfaces for cell attachment and growth.
One way to guide the cells is via spatial control of the scaffold's properties. Our research is directed at incorporating instructions for the cells into tissue engineering scaffolds by patterning architecture and surface chemistry, two key factors governing the interactions of cells and tissues with a scaffold. For example, tissue engineering devices can be composed of large channels (>100 μm) as well as small pores to allow infiltration of tissues such as blood vessels, as well as provide surface area for cell attachment and growth [1], [2]. And, patterned surface chemistry can provide chemical “instructions” to cells about where to attach, where to migrate to, and/or how to differentiate.
The goals of the experiments in this paper were twofold: to demonstrate that patterning of cell behavior in three-dimensional scaffolds is possible, and to begin to collect data on cell response to different surface chemistries in this patternable system. The ultimate goal will be to collect data on cell responses to many patternable factors, and to use this data to design improved tissue engineering devices.
Scaffolds were constructed using the Three-Dimensional Printing™(3DP™) process, which can create three-dimensional patterns of composition and architecture (at a resolution of ∼100 μm for this system) [3], [4], [5], [6], [7]. Three-dimensional parts are constructed by sequentially printing layers, each of which binds to the preceding layer. Surface chemistry and bulk properties are selectively modified by printing surfactants and other molecules only in desired locations. Porosity is created by mixing salt with the initial polymer powder and leaching the salt from the device after fabrication.
Our material system was chosen based on the previous work of Park et al. [8] to pattern fibroblast and hepatocyte cell adhesion on two-dimensional surfaces. Their approach was to first block non-specific cell adhesion to surfaces by enriching the surfaces with poly(ethylene oxide) (PEO), and then to tether ligands to the PEO molecules to encourage adhesion of specific cell types [9], [10]. They explored a variety of polymers and PEO surfactants, and we chose to use the combination which showed the most striking patterning on 2D surfaces: poly(l-lactide-co-glycolide) (PLLGA) and Pluronic® F-127. Pluronic® molecules are A-B-A tri-block copolymers with a hydrophobic poly(propylene oxide) (PPO) block sandwiched between two PEO blocks. The PPO chain will adsorb to hydrophobic surfaces or intermingle with dissolved PLLGA polymer chains during fabrication to anchoring the Pluronic® to a surface. We explored the effect of surface chemistry on cell response by incorporating differing amounts of PEO into PLLGA scaffolds during fabrication.
The specific application focus of this paper was bone tissue engineering. Several cell behaviors that are important in bone wound healing are adhesion, growth, migration, and differentiation. Four types of assays were performed to assess the effect of PEO concentration on these cell functions: cell adhesion, invasion assay, alkaline phosphatase (ALP) activity, and observation by SEM. ALP activity is a common assay for bone cell response to surfaces; the hypothesis is that elevated ALP levels indicate that the surface influences the bone cells to differentiate [11], [12], [13], [14], [15], [16], [17]. The invasion assay is based on surrounding a scaffold with a reservoir of cells and quantifying how many enter the scaffold [18]. Because of the length of the experiment, the invasion assay measures a combination of migration and proliferation.
Section snippets
Fabrication of polymer substrates
After fabrication, all substrates were dried on a lyophilizer (<25 μTorr) for at least 48 h before use.
Materials: Panacryl® PLLGA (Mw ∼300,000; 95:5 lactide:glycolide monomer ratio) was received as a gift from Ethicon, Inc. in the form of 8 mg pellets. Panacryl® pellets were milled cryogenically and then sieved to a particle size range of 78–150 μm. NaCl (VWR) was milled at room conditions and sieved to a particle size range of 125–150 μm. Pluronic® F-127 (Mw ∼12,500 Da, nominally 98-67-98
Adhesion of MG-63 cells on patterned 3DP™ surfaces and scaffolds
The ability of 3DP™ fabricated devices to create pattern cell responses was tested by creating two devices striped with PEO modified regions. The devices were collagen coated, seeded with MG-63 cells at 100,000 cells/cm2, and incubated for 24 h.
Fig. 1 is a photomigraph of a “flat” 3DP™ fabricated surface printed with three 1 mm stripes of differing PEO concentrations (the first stripe was misprinted). The cells are stained dark blue and the PEO regions are plainly visible as the lighter vertical
Conclusions
The focus of this paper was the assessment of cell response to different surface chemistry modifications of 3D scaffolds and an investigation of patterning in three dimensions. Our system was osteoblast response to scaffolds fabricated using the 3DP™ process from PLLGA scaffolds and PEO.
We demonstrated patterning of cell adhesion on 3DP™ fabricated ‘flat’ surfaces and porous scaffolds, both fabricated with stripes of PEO. Unfortunately, the results were not as striking as patterning on solvent
Acknowledgements
This work was supported by Therics, Inc., an NSF Graduate Fellowship, and an NIH/NIGMS Biotechnology Training Grant to MIT.
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