Introduction

It is becoming increasingly apparent that the surface on which cells are cultured in vitro is an important factor in their adherence and subsequent growth. Most in vitro health science studies rely on cell culture methodologies to carry out investigations of eventual clinical importance (i.e., biopharmaceuticals intended for therapies or biomaterials designed for medical devices). For example, culturing cells such as monocyte-derived macrophages (MDMs) has become very important to studies on inflammatory responses, particularly when investigating the foreign body reaction to various biomaterials. Previous studies have shown that material surface chemistry can modulate MDM behavior, including cell spreading, fusion to foreign body giant cells (FBGCs), and degradative capabilities (1).

Tissue culture polystyrene (TCPS) is the most commonly used in vitro cell culture surface (2)3, and polydimethylsiloxane (PDMS) has been used as a primary reference material for the evaluation of new biomaterials (4). However, very few studies have fully characterized basic cell functions that may be affected by these standard control surface chemistries as they evaluate parameters of interest between test substances and cells in culture (5,6,7), thereby justifying the importance for a study of this nature.

Research activity in the area of tissue regeneration is quickly gaining interest. Many of the early screening studies for biomaterials used in tissue engineering or wound repair are first tested on TCPS or PDMS culture ware or enclosures. This raises the question: what perturbations in cell response are introduced as a result of these artificial substrates, and do they contribute to conclusions regarding new test materials? For example, a significant influence of cell culture vessels on the assessment of degradable polyurethanes developed in the biomaterials field was reported in a recent study with degradable polycarbonate-based polyurethanes (PCNUs) with identification of products [such as methylene dianiline (MDA) and butanediol (BD)] that could confound the results (8). Given the importance of products in cell response during polyurethane biodegradation demonstrated by a previous study (9), nondegradable polymers have been and will continue to be used for the study of baseline cellular responses to various exogenous stimuli.

Previously, studies have shown that degradable PCNUs adversely influenced MDM morphology and function (10). Based on those results, it was of interest to compare the effect of two nondegradable surfaces (TCPS and PDMS), which could be used during in vitro testing of biodegradable polymers using the same parameters. In this study, the functional responses of differentiated U937 cells, previously established as a reproducible cell model for studying cell-material interaction (11), were measured in terms of esterase and acid phosphatase (AP) activity as well as protein levels and de novo proteins released. In addition, the relative values of the above three degradable materials were compared with the response of the nondegradable standard control surfaces.

Materials and Methods

Unless otherwise specified, all reagents were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada).

Preparation of Material Surfaces

The three model PCNUs used in this study were synthesized as described previously either with hexane diisocyanate (HDI) or 4,4′-methylene bis phenyl diisocyanate (MDI); a 1,6-hexyl polycarbonate diol soft segment (PCN); and a 1,4-BD chain extender in stoichiometric ratios of either 4:3:1 or 3:2:1 (12). A ¹⁴C radiolabel was incorporated into the PCNU structure with BD as a method to assess biodegradation for HDI321 and MDI321, whereas HDI431 had the radiolabel (¹⁴C) incorporated into the HDI moiety. Previously it was shown that the position of the radiolabel did not affect the amount of degradation (13). The PCNUs were dissolved overnight in a 10% dimethylacetamide solution (w/v), filtered (0.45-µm Teflon® filter; Chromatographic Specialties, Toronto, ON, Canada), and then coated as a thin layer onto 15-mm diameter glass coverslips (Fisher Scientific Ltd., Ottawa, ON, Canada) under sterile conditions as previously described (9).

U937 Cell Line

U937 cells were maintained and differentiated for 72 h with phorbol myristate acetate (PMA; 1 × 10^-7 M) as previously described (11). After the differentiation period, the medium was replaced in each well, and the cells were mechanically disrupted prior to reseeding at a density of 1.0 × 10⁶ cells/well on TCPS coverslips (Sarstedt, Newton, NC, USA), medical grade PDMS films (0.01” thick, Specialty Manufacturing, Saginaw, MI, USA), or coverslips coated with HDI431, HDI321, or MDI321 in 24-well tissue culture plates. After an incubation period of 1 h, the growth medium was replaced to eliminate any nonadherent cells, and the cell material interaction was continued for 48 h.