286086 Effects of PLGA Nano Patterns of Various Dimensions On Osteoblast and Osteosarcoma Cell Responses
Effects of PLGA Nano Patterns of Various Dimensions on Osteoblast and Osteosarcoma Cell Responses
Yongchen Wang1, Lijuan Zhang1, Linlin Sun2, and Thomas J. Webster2
1Department of Chemistry, Brown University, Rhode Island 02912, USA
2School of Engineering, Brown University, Rhode Island 02912, USA
Abstract: The interactions between substrates and cells, and especially the use of nanotopographies on the substrates to manipulate cell functions have been widely studied. Among these studies, research on the effects of nanotopographies on cancer cell behaviors can help develop new strategies for cancer therapy. In this work, nano patterns were fabricated on a biocompatible and biodegradable co-polymer, poly(lactic-co-glycolic acid) (PLGA), with a template method 1, 2. The sizes of nano patterns included 27nm, 190nm, 300nm, 400nm, and 520nm, and a flat surface feature was also made as a control. The surface features, root-mean-square (RMS) roughness and wettability of PLGA nano patterns were characterized. Both human healthy osteoblasts (OB) and human cancerous osteosarcoma cells (OS) were cultured on these PLGA surface features and the effects of PLGA nano patterns on their cell adhesion was evaluated by 4 hour cell adhesion assays. In the cell adhesion assays, 27nm PLGA nano patterns enhanced both osteoblast and osteosarcoma cell adhesion the most compared with other surface features and there were relatively strong correlations between either osteoblast density or osteosarcoma cell density and the RMS roughness of PLGA nano patterns. More interestingly, 27nm PLGA nano patterns exhibited a significant increase in osteoblast density compared with osteosarcoma cell density cultured on the same surface features and therefore could be further developed for a novel bone cancer therapy instead of the traditional cancer therapy methods.
Experiments: The nano patterns were initially created by the self-assembly of polystyrene (PS) beads of 27nm, 190nm, 300nm, 400nm, and 520nm. These patterns were first transferred from PS beads to polydimethylsiloxane (PDMS) molds, which polymerized in close contact with the PS beads, and then were transferred from PDMS molds to PLGA films, which formed in close contact with the PDMS molds after solvent volatilization. After synthesis, the surface features of PGLA nano patterns were studied by AFM. RMS roughness values were calculated from flattened z-sensor scan AFM images and the wettability were characterized with a contact angle goniometer to measure the water contact angles of the PLGA nano patterns.
Human healthy osteoblasts (CRL-11372, ATCC) and human cancerous osteosarcoma cells (CRL-1427, ATCC) were cultured in their respective complete culture medium under a standard environment. The cell adhesion of both osteoblasts and osteosarcoma cells were evaluated by culturing these two types of cells on the PLGA nano patterns for 4 hours and investigating the cell density by means of MTT assays. Cell numbers were counted with a hemocytometer. All cell studies were accomplished in triplicate and the data were analyzed with a one-tailed student t-test.
Results and Discussion: From height scan AFM images (Scanning size: 5µm°Á5µm), nano patterns of 27nm, 190nm, 300nm, 400nm and 520nm and a flat PLGA surface were fabricated (Fig. 1). The RMS roughness and wettability of different PLGA surface features were characterized (Table. 1). 27nm PLGA nano patterns exhibited the largest RMS roughness and its surface was the most hydrophobic. Flat PLGA surfaces showed the smallest RMS roughness and its surface was the most hydrophilic.
Figure. 1. AFM images of PLGA films with (A) flat surfaces; (B) 27nm; (C) 190nm; (D) 300nm; (E) 400nm; and (F) 520nm nano patterns.
Table. 1. RMS roughness and water contact angles of the flat PLGA film and PLGA films with 27nm, 190nm, 300nm, 400nm, and 520nm nano patterns.
For osteoblasts alone or osteosarcoma cells alone, the 27nm nano patterns increased cell adhesion the most (Fig. 2). For osteoblasts and osteosarcoma cells cultured on the same surface feature, only 27nm nano patterns showed a significant osteoblast cell density increase compared with osteosarcoma cell density. From the correlation plots between cell density of osteoblasts or osteosarcoma cells, and the physical properties including RMS roughness and wettability (Fig. 3; Table. 2), there were relatively strong correlations between either osteoblast density or osteosarcoma cell density and the RMS roughness of PLGA nano patterns, and relatively weak correlations between either osteoblast density or osteosarcoma cell density and the wettability of PLGA nano patterns. These results could be explained in respect to several aspects. First, larger RMS roughness means larger surface area and more sites for the adsorption of proteins regulating cell adhesion. Second, the RMS roughness of 27nm PLGA nano patterns were the closest to the roughness of the real bone matrix 3. Third, 27nm is the closest to the dimensions of the extracellular matrix proteins participating in cell adhesion 4, 5. However, currently, the mechanism of the effects of PLGA nano patterns on the cellular responses of osteoblasts and osteosarcoma cells is not known. Further study is needed to fully understand this.
Figure. 2. 4 hr cell adhesion results of OB and OS (N=3; seeding density: 3500 cells/cm2). The cell densities are average values +/- S.E.M. For OB alone, *, ** and *** represents p<0.05 compared with the flat, 27nm and 300nm surface features, respectively; for OS alone, ^, ^^, ^^^ and ^^^^ represents p<0.05 compared with the flat, 27nm, 190nm and 300nm surface features, respectively; and + represents p<0.05 for OS cell density compared with OB density on the same surface features.
Figure. 3. Correlation plots between osteoblast density/RMS values obtained from AFM, osteoblast density/ contact angle, osteosarcoma cell density/RMS values obtained from AFM and osteoblast cell density/contact angle.
Table. 2. R2 values and R values of the correlation plots between osteoblast density/RMS values obtained from AFM, osteoblast density/contact angle, osteosarcoma cell density/RMS values obtained from AFM, and osteosarcoma cell density/contact angle.
Conclusions: PLGA nano patterns of various dimensions could be fabricated with a template method. For cell studies, 27nm PLGA nano patterns not only increased the cell density most for osteoblasts alone or osteosarcoma cells alone, but it also significantly increased osteoblast density compared with osteosarcoma cell density cultured on the same surface features. It was also found that there were relatively strong correlations between either osteoblast or osteosarcoma cell densities, and RMS roughness of PLGA nano patterns. Thus, 27nm PLGA nano patterns should be further studied as a potentially novel bone cancer therapy with few adverse effects.
Acknowledgements: We would like to thank Hermman Foundation for their financial support, and Vera Fonseca and Professor Eric Darling for their technical support with AFM use.
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