Ann Thorac Surg 2001;71:S437-S440
© 2001 The Society of Thoracic Surgeons
Basic research
Surface attached ultrathin polymer monolayers for control of cell adhesion
Manfred Dahm, MD, PhDa,
Bung-Jun Chang, MSa,
Oswald Prucker, PhDa,
Melanie Pierkes, MDa,
Tatjana Alt, BSa,
Eckhard Mayer, MD, PhDa,
Jürgen Rühe, PhDa,
Hellmut Oelert, MD, PhDa
a Department of Cardiothoracic and Vascular Surgery, University-Hospital of Mainz, Mainz, Germany
Address reprint requests to Dr Dahm, Department of Cardiothoracic and Vascular Surgery, University-Hospital Mainz, Langenbeckstrasse 1, 55131 Mainz, Germany
e-mail: mdahm{at}mail.uni-mainz.de
Presented at the VIII International Symposium on Cardiac Bioprostheses, Cancun, Mexico, Nov 3–5, 2000.
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Abstract
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Background. Calcific degeneration is the major drawback of bioprostheses. None of the numerous preventive approaches omitted calcification. Previous studies showed that cellular surface seeding decreases calcium uptake in vitro but achievement of coverage remains problematic. A new approach is presented masking glutaraldehyde residues with a polymer layer allowing cell seeding. The aim of this study was to evaluate different polymers for suitability.
Methods. Ten polymers—covalently bound to glass—were tested for their ability to seed animal and human cells. Quality of coverage was evaluated by light and scanning electron microscopy, and polymers were characterized physicochemically.
Results. Quality of cellular growth was similar for canine and human cells. Five polymers allowed excellent surface coverage, two led to a decrease of cell adherence, and four to poor cellular growth. No correlation between molecular weight, thickness, hydrophilicity, or charge of the polymer and cell growth was found.
Conclusions. Polymer monolayers can promote cellular growth but without correlation to physicochemical characteristics. Polymers covalently bound to biologic tissue appear to be a promising approach for achieving cellular coverage of biomaterials.
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Introduction
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Calcific tissue degeneration with the resulting need for reoperation remains the major drawback of bioprostheses [1, 2]. Pathophysiology of tissue failure is multifactorial, and some aspects still remain unclear.
Various approaches applying chemical treatments to prevent calcium binding have been proposed, but none has been shown to omit calcification [3–5]. Strategies aimed at reducing binding sites for calcium either blocked binding sites or, recently, addressed glutaraldehyde remnants as a source for calcifications. Despite evidence that glutaraldehyde plays a major role in the calcification process, most of the currently applied tanning protocols for biologic materials still include the use of glutaraldehyde. Aldehyde treatment increases mechanical strength of the implant material and prevents shrinking, but as a negative effect, toxic residues prevent reendothelialization [6]. In previous studies we and others have shown that surface seeding with cells decreases calcium uptake in vitro [7], but achievement of complete cellular coverage remained a substantial problem [8].
This difficulty has stimulated us to develop a new concept: we did not intend to modify the tanning process as good mechanical properties of glutaraldehyde-tanned biologic tissues have been documented during the last 30 years, but aimed to mask glutaraldehyde residues by an ultrathin layer of a hydrophilic polymer covalently bound to the surface of the biologic tissue. Second, vital cells are seeded on top. The aim of this model studies was to evaluate several polymers for their suitability to build a covalently bound monolayer and allow cellular coverage.
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Material and methods
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Polymers
Ten hydrophilic polymers [poly(dimethyl acrylamide); polyacrylic acid; polyacrylamide; poly(2-hydroxyethyl methacrylate); polyvinyl alcohol; poly(2-ethyl-2-oxazoline); chitosan; poly(2-[methacryloyloxy]-ethyltrimethylammonium chloride); polyethylenimine; poly(methacrylic acid); structure of the polymers is given in Figure 1, were immobilized on glass slides using a photochemical approach: monolayers carrying benzophenone units were first deposited on the surface and then covered with polymer coatings [9]. On irradiation with ultraviolet light, the benzophenone moieties react through a radical coupling reaction with alkyl C-H groups and attach the chain to the surface.
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Fig 1. Structure of the tested hydrophilic polymers. Insert displays the benzophenone-mediated binding of the polymer to the modified glass surface.
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Molecular weight of the polymers was determined by light scattering, thickness by ellipsometry on reference layers on silicon wafers, and hydrophilicity by measuring water contact angles of the bulk and monolayers.
Cell growth studies
All specimens were tested for their ability to seed and grow cells (rat [2] and rabbit fibrocytes [2]; human endothelial cells [3]). Cell growth was examined on 1 x 1 cm2 samples of the polymers on glass; cell growth on noncoated glass slides served as a control. Cells (5 x 104) were placed on each sample and cultured for 2 weeks (CO2, 37°C; canine cells: RPMI 1640 with L-glutamine, 10% fetal calf serum, 1% penicillium/streptomycin, 1% nonessential amino acids, 1% sodium pyruvate; human endothelial cells: endothelial cell growth medium, supplemented with 2% fetal calf serum, 0.4% endothelial cell growth serum, 0.1 ng/mL epidermal growth factor, 1 ng/mL basic fibroblast growth factor, 1 mg/mL hydrocortisone, 50 µg/ml gentamicin/50 mg/ml amphotericin) on six-well plates (Greiner, Frickenhausen, Germany). Every 2 days half of the medium was replaced, all samples were examined by phase-contrast microscopy, and the progress of cellular coverage was documented photographically using the following score (Table 1): 1 = excellent growth (comparable to glass reference), 2 = growth less than on reference, 3 = less than 50% of surface covered or small agglomerates, 4 = single cells or abnormal morphology).
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Table 1. Physicochemical Properties of Tested Polymers and Seeding Behavior of Different Cell Lines According to Microscopic Appearance of Cell Layer
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In addition, after the 14 days study period one representative specimen from each cell line and polymer was prepared for scanning electron microscopy.
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Results
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Despite the fact that all polymers used in this study are water-soluble—except for poly(2-hydroxyethyl methacrylate) and chitosan, which absorb water —physicochemical properties differed greatly among the tested polymers. Molecular weight, thickness, contact angles of bulk and surface layer, and charge for the tested polymers are shown on Table 1.
Range of molecular weights was between 30,000 and 5,800,000 g/mol. All layers were ultrathin with a maximum thickness of 26.2 nm and minimum thickness of 2.5 nm, and generally proportional to the molecular size (molecular weight) of the polymers. Contact angles for bulk and surface layer were in a wide range but mostly less than 50 degrees, indicating the hydrophilicity of the bulk and surface layer. Values obtained from the monolayers were higher than those from the bulk owing to the fact that all layers swell in contact with water. Charge of the polymers was neutral in seven, positive in two, and negative in two polymers.
Serial photo documentation of growth and extent of cellular coverage (Fig 2) revealed different behavior of the polymers: five (polyethylenimine, poly(methacrylic acid), chitosan, poly(2-[methacryloyloxy]-ethyltrimethylammonium chloride), poly(2-ethyl-2-oxazoline); (Figs 2A, 2B) allowed excellent seeding with complete cell coverage during the study period comparable to the controls, two polymers showed a decrease of cell adherence after 1 week (chitosan, polyvinyl alcohol). Four polymers led to poor cellular coverage (poly(dimethyl acrylamide), polyacrylic acid, polyacrylamide, poly(2-hydroxyethyl methacrylate); (Figs 2C, 2D), and one (polyvinyl alcohol) showed varying results with the different cell types (Table 1). Scanning electron microscopy confirmed these results, showing an intact cellular layer on those five polymers that promoted cell growth.
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Fig 2. Phase contrast pictures of poly(2-ethyl-2-oxazoline) (PetOx) and poly (methacrylic acid) (PMAA) with excellent growth and complete coverage of the surface by human endothelial cells (A) and sheep cells (B) (x10). Extremely poor cell growth of rat (C) (x20) and human endothelial cells (D) on polyacrylamide showing that the cells lost their integrity. No coverage was achieved (D) (x20).
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No correlation between the achievement of complete cell coverage of the surface and physicochemical properties of the polymers was found.
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Comment
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Shortly after the introduction of untreated biologic implants, shrinking and tearing of the tissue was reported. The use of glutaraldehyde for presevation of the material significantly improved the mechanical stability of the implants, leading to better mid-term and long-term results. Aldehyde cytotoxicity appears to be an important disadvantage of the tanning process as it transforms the biologic tissue into a nonvital implant prone to plasma insudation and calcium accumulation. Seeding of the surface of the decellularized surface with vital host cells to recreate a biologic barrier seemed an intriguing concept to improve long-term results. Beneficial effects of cell coverage on calcium uptake have been demonstrated in vitro and in vivo [7], but achievement of complete cellular coverage proved to be the major problem.
This has stimulated research for the creation of totally vital implants. Using tissue engineering techniques, impressive results were obtained: degradable polymer meshes or decellularized biologic structures were seeded with or replaced by vital cells [10–12], but concern occurred for the function of these implants under high-pressure conditions. So far, only studies with implanted valves in the pulmonary circulation have been reported [12].
The concept proposed in this article is more conservative: it combines the use of glutaraldehyde-tanned biologic material—which has proven to perform satisfactorily in the human circulatory system—with the implementation of a new approach to achieve cellular coverage. Aldehyde residuals that remain after the glutaraldehyde tanning process are masked with a covalently bound polymer. In a second step, this polymer is seeded with cells.
Polymers are chemical substances covering a wide range of properties. Hydrophilic polymers appear suitable to promote cell growth but are unstable in aqueous environments like the bloodstream. This problem was solved with the reported chemical approach, and stable covalent bindings to glass and collagen were achieved.
The results from the cell-seeding studies reveal that cells can grow on some polymers and form a dense layer. The results are very consistent for different cell types including endothelial cells. The additional information provided by measuring calcein uptake shows that these cells maintain their integrity during the study period.
However, it has not been possible to characterize physicochemical properties that allow excellent cell growth. Tamada and Ikada [13] claimed that rat fibroblasts grow best on substrates with contact angles of approximately 70 degrees, a finding we cannot support from our study. Because we could not evaluate a correlation with physicochemical characteristics, careful testing of each polymer seems mandatory, especially as vital cells may behave differently in adhesion and proliferation with slight modifications of side chains of polymers.
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Acknowledgments
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Supported by the German Research Foundation, Da246/3–1.
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References
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Abstract
Introduction
