Grafting of bovine serum albumin proteins on plasma-modified polymers for potential application in tissue engineering
© Kasálková et al.; licensee Springer. 2014
Received: 12 December 2013
Accepted: 16 March 2014
Published: 4 April 2014
In this work, an influence of bovine serum albumin proteins grafting on the surface properties of plasma-treated polyethylene and poly-l-lactic acid was studied. The interaction of the vascular smooth muscle cells with the modified polymer surface was determined. The surface properties were characterized by X-ray photoelectron spectroscopy, atomic force microscopy, nano-LC-ESI-Q-TOF mass spectrometry, electrokinetic analysis, and goniometry. One of the motivations for this work is the idea that by the interaction of the cell with substrate surface, the proteins will form an interlayer between the cell and the substrate. It was proven that when interacting with the plasma-treated high-density polyethylene and poly-l-lactic acid, the bovine serum albumin protein is grafted on the polymer surface. Since the proteins are bonded to the substrate surface, they can stimulate cell adhesion and proliferation.
KeywordsPolymers Plasma treatments Protein grafting Surface characterization Cell interaction
Tissue engineering (TE) is the discipline which includes both creation of the new tissue and design and realization of the cells on substrates [1, 2]. Substrates play a key role in creation of the cell environment . To guide the organization, growth, and differentiation of cells in TE constructs, the biomaterial scaffold should be able to provide not only a physical support but also the chemical and biological clues needed in forming functional tissue [4–6].
Biomaterials and various synthetic and natural materials, such as polymers, ceramics, metals, or their composites, have been investigated and used in different manners [5, 7]. Polymeric materials have been widely studied as substrates for tissue engineering due to their unique features such as mechanical properties, high availability, low cost, and relatively easy design and production [6, 8]. However, only a few polymers provide the biocompatibility needed to be used with the cells in vitro and in vivo. High-density polyethylene (HDPE) has been extensively used for application such as the part of orthopedic implants . To induce a regeneration process and to avoid the problems due to tissue replacement with a permanent implant, research has been oriented towards the development of polymers that would degrade and could be replaced by human tissue produced by the cells surrounding the material . Despite of their advantages, however, some of their characteristic properties, like wettability, adhesion, surface composition, and suchlike are insufficient for many applications. The positive effect of the above-mentioned properties and also biocompatibility of the polymer surface provide an opportunity of modification of existing material with bioactive molecules (amino acids, peptides, anticoagulants) bound by covalent bonds to polymer surface [11–13].
Polymer surfaces are often modified by thin layers of protein-like collagen or fibronectin to improve their biocompatibility . Bioactive molecules influence also the growth factors and regulate cell adhesion, migration, and proliferation [9, 15]. Bovine serum albumin (BSA) is a globular protein that is used in numerous biochemical applications. Bovine serum albumin (BSA) can be used as a reference (model) protein in which its properties are compared with other proteins. BSA is also included in the protein part of the various media used for operations with cells. BSA was chosen as a representative protein present in cell culture as a supplement to increase the growth and productivity of cells and increase overall cell health.
A very important part of the general study of biocompatibility of materials is the surface characterization of the prepared substrates and adhered bioactive compounds. As basic parameters influencing the cell-substrate interaction, surface chemistry, polarity, wettability morphology, and roughness can be included.
In this work, the influence of BSA protein grafting on the surface properties of the polyethylene (HDPE) and poly-l-lactide acid (PLLA) was studied. HDPE was chosen as the representative of the non-polar/non-biodegradable polymer. With its very simple structure containing only carbon and hydrogen atoms, this polymer can serve as a model material. PLLA was chosen as a polar/biodegradable polymer, whose cell affinity is often compromised because of its hydrophobicity and low surface energy . The surface properties were characterized by X-ray photoelectron spectroscopy, nano-LC-ESI-Q-TOF mass spectrometry, atomic force microscopy, electrokinetic analysis, and goniometry. One of the motivations for this work is the idea that due to cell interaction with the substrate, the proteins will form an interlayer between the cell and the substrate surface .
Materials and chemical modification
The experiments were performed on HDPE foil (thickness 40 μm, density 0.951 g cm−3, Granitol a.s. CR, Moravský Beroun, Czech Republic) and biopolymer PLLA foil (50 μm, 1.25 g cm−3, Goodfellow Ltd., Huntingdon, UK).
The surface modification of polymer substrates consisted of plasma treatment and subsequent grafting with proteins. The samples were modified by plasma discharge on Balzers SCD 050 device (BalTec Maschinenbau AG, Pfäffikon, Switzerland). The parameters of the deposition were DC Ar plasma, gas purity 99.995%, flow 0.3 l s−1, pressure 8 Pa, power 3 W, electrode distance of 50 mm, and time 300 s.
Immediately after treatment, the activated polymer surface was grafted by immersion into water solution of BSA (concentration 2 wt.%, Sigma-Aldrich Corporation, St. Louis, MO, USA) for 24 h at room temperature (RT). The excess of non-bound molecules was removed by consequent immersion of the samples into distilled water for 24 h. The samples were dried at RT for 13 h.
The surface wettability was determined by water contact angle (WCA) measurement immediately after modification and after 17 days using distilled water (drop of volume 8 μl) at 20 different positions and surface energy evaluation system (Advex Instruments, Brno, Czech Republic). WCA of the plasma-treated samples strongly depends on the time from treatment.
The presence of the grafted protein molecules on the modified surface was detected by nano-LC-ESI-Q-TOF mass spectrometry. The samples were placed in Petri dish, and 10 μl of solutions (2 μl trypsin, concentration 20 μg μl−1 in 100 μl 50 mmol l−1 NH4HCO3) was applied on the sample surface. In the inside perimeter of Petri dishes, pieces of wet pulp were placed, in order to avoid drying of the solution on the surface of foils, and consequently the dish was closed. After 2 h of the molecule cleavage, new peptides were concentrated and desalted by reverse-phase zip-tip C18 (EMD Millipore Corporation, Billerica, MA, USA) at RT.
The presence of the carbon, oxygen, and nitrogen atoms in the modified surface layer was detected by X-ray photoelectron spectroscopy (XPS). The spectra of samples were measured with Omicron Nanotechnology ESCAProbeP spectrometer (Omicron Nanotechnology GmbH, Taunusstein, Germany) (1,486.7 eV, step size 0.05 eV, area 2 × 3 mm2). This elemental analysis was performed 17 days after modification of the samples.
The changes in surface morphology and roughness of samples were examined 17 days after modification by atomic force microscopy (AFM) using a Veeco CP II device (Bruker Corporation CP-II, Santa Barbara, CA, USA) (‘tapping’ mode, probe RTESPA-CP, spring constant 20 to 80 N∙m−1). The surface roughness value (Ra) represents the arithmetic average of the deviation from the center plane of the samples.
The electrokinetic analysis (zeta potential) of the samples was done using SurPASS instrument (Anton Paar, Graz, Austria), (adjustable gap cell, 0.001 mol∙dm−3 electrolyte KCl, pH = 6.3, RT). The values of the zeta potential were determined by two methods, a streaming current and a streaming potential and calculated by Helmholtz-Smoluchowski and Fairbrother-Mastins equations . Each sample was measured four times with the experimental error of 10%.
Biological test of adhesion and proliferation
For evaluation of cell number and morphology in cell culture experiments, three pristine and modified HDPE and PLLA samples were used for analysis by randomly chosen fields. The samples were sterilized for 1 h with 70% ethanol, air-dried in a sterile environment to prevent possible negative effects of alcohol on the cells, and inserted into 12-well plates (TPP, well diameter 2 mm). Samples were seeded with smooth vascular muscle cells (VSMCs) derived from rat aorta by an explantation method (passage 7). VSMCs were seeded with the density 17,000 cells/cm2 into 3 ml of Dulbecco's modified Eagle's minimum essential medium (DMEM, Sigma) supplement with 10% fetal bovine serum (FBS, Sebak GmbH, Aidenbach, Germany). The cells were cultivated for 2, 4, and 6 days at 37°C in a humidified air atmosphere containing 5% CO2.
On the 2nd, 4th, and 6th day after seeding, the cells were rinsed in phosphate buffered saline (PBS) and fixed for 1 h in 70% cold ethanol (−20°C). The samples used for analysis by randomly chosen field were stained for 40 min with a combination of fluorescent membrane dye Texas Red C2-maleimide (Molecular probes, Invitrogen, Carlsbad, CA, USA) and a nuclear dye Hoechst no 33342 (Sigma). The number, morphology, and distribution of cells on substrate surface were then evaluated on photographs taken under an Olympus I×51 microscope using an Olympus DP 70 digital camera (Olympus America Inc., Center Valley, PA, USA). The number of cells was determined using image analysis software NIS Elements (Nikon Instruments Inc., Melville, NY, USA).
Results and discussion
Physical and chemical properties
Peptides detected on the surface of grafted HDPE and PLLA proved using mass spectrometry
Fibrinogen alpha chain
Fibrinogen alpha chain
Atomic concentration of selected elements determined in surface layer of polymers using XPS
Atomic concentration (%)
Cell adhesion, growth, and proliferation
Number of VSMCs (cells/cm 2 ) cultivated 2, 4, and 6 days on HDPE and PLLA
Number of VSMCs (cells/cm2) cultivated
The explanation of biocompatibility improvement of surface after plasma modification and protein grafting is connected with surface chemistry change, especially with amino groups presented on the modified surface. It is known that the major proteins (especially proteins of fetal bovine serum) as well as cell membranes are negatively charged under physiological pH. The adhesion of cells with negatively charged membranes may be facilitated by electrostatic interactions and the better cell adhesion may be expected on positively charged surfaces [20–22]. The surface charge (of solid substrates and of cells) significantly determines both cell-cell and cell-solid interactions. In low ionic strength environment, the adhesion is influenced mostly by electrostatic interactions between surfaces, where the surface chemistry, surface functional groups, and surface charge play the important role; while in increasing ionic strength (increasing concentration of surroundings), the importance of non-polar (hydrophobic) interactions grows . Also, it was presented earlier for human umbilical vein endothelial cells  or for human fibroblasts  that better protein adsorption occurs if the surface contains -NH2 groups. Adsorbed proteins play a major role in the attachment of anchorage-dependent cells through their binding to integrins .
These results are contrary to the majority of theories, in which albumin is considered a non-adhesive molecule. But albumin can support of the adsorption of some molecules (like vitronectin or fibronectin) from the culture serum and thus can indirectly and positively influence cell's adhesion and proliferation. The molecules may be synthesized and deposited by VSMCs and may cause the increase of the cell's activity .
It was proven that during interaction of BSA protein with the plasma-treated polyethylene and poly-l-lactic acid, BSA protein is grafted on their surfaces. Chemically bonded BSA protein was confirmed by XPS, mass spectrometry, AFM, electrokinetic analysis, and goniometry. This result is a significant contribution to the understanding of cell and substrate behavior during cell interaction with chemically active polymer in tissue engineering field. Due to plasma treatment and subsequent BSA grafting to polymer surface, the cell adhesion and proliferation can be stimulated due to the presence of active functional groups on the surface, which improves the electrostatic interactions between substrates and cells.
atomic force microscopy
bovine serum albumin
vascular smooth muscle cell
water contact angle
X-ray photoelectron spectroscopy
This work was supported by the GACR under project P108/12/G108.
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