Biofunctionalization of surfaces by covalent co-immobilization of RGD and PHSRN-containing oligopeptides

Surface functionalization of biomedical materials provides materials with specific biological activities. This is relevant to the implant industry in the design and modification of implant surfaces in a way conducive to tissue regeneration and accelerating the healing process.

Titanium (Ti) is a material often used for the construction of dental and orthopedic implants, because of its excellent mechanical characteristics, chemical stability, and biocompatibility [1]. However, because titanium is an inert material, and as such cannot encourage cell growth on and around the implant site, titanium implants may hinder bone formation [2]. Another problem, which similarly impacts the healing process, is infections at the implantat site [3,4].

One of the methods used to try and overcome these medical problems is functionalization of the titanium surfaces by coatings the implant with materials containing molecules with known biological activities [5,6]. A variety of molecules have been used in recent years to coat Ti surfaces, including bioactive proteins (Bone Morphogenic Proteins (BMPs), collagen) [7-10], nucleotides [11,12], peptides (RGD, FNIII [7-10]) [13-17]), and antimicrobial agents [18-22], although presently molecules must be coated individually. The next challenge in implant coating technology will be coating the same surface with multiple materials that will contribute to a variety of surface bioactivities, including mineral formation, cell recruitment, cell differentiation and proliferation, improvement of osseointegration, and antimicrobial activities; in order to do that, many factors must be explored, such as electrostatic interactions and physical absorption of the different molecules that are used to coat the Ti surface [23]. This research has lead to promising coating techniques, such as hyaluronic acid/chitosan polyelectrolyte multilayer combined with RGD peptides, which inhibits bacterial adhesion and growth and promotes bone cell functions [24]. Another example is a Ti surface bearing BMP-2 and gentamicin, which enhances osseointegration, combined with antibacterial properties [25].

A major problem arises regarding the implant coating stability. Physical absorption and electrostatic attraction, for example, may not be suitable methods for producing coatings that are stable enough to remain functional after surgical scenarios or in the biological environment around the implantation site. A coating technique with promising results is coating by covalent bonding between the Ti and the molecules. Methods for biomechanical immobilization, such as coupling with silane agents [16,26] or thiols [27,28], were shown to produce surfaces with strong mechanical and biomechanical stability.

In the 2013 article “Surface biofunctionalization by covalent co-immobilization of Oligopeptides” [29], Chen et al present a simple and reliable oligopeptide coating method using covalent conjugation to co-immobilize two oligopeptides known for their cooperative bioactivities: PHSRN and RGD. While PHSRN is not biologically active by itself, it has a synergistic effect with RGD, as these two molecules cooperate in the construction of fibronectin [30,31]. Recombinant peptides, which include motifs from these two peptides [32-36], and multi-peptide systems including PHSRN and RGD [31], were shown to increase cell adhesion, proliferation, and expansion, in comparison to systems that use the RGD motif only. In their study, Chen et al incorporated peptides that contain PHSRN and RGD motifs onto Ti surfaces, using organosilanes for covalent linking. The synergistic effect on cell response of these two peptides was tested on model surfaces, and the ability of these modified Ti surfaces to retain the biological characteristics of the immobilized peptides was explored.

The oligopeptides described in the article were attached to the surface in a three-step method using CPTES as a coupling agent to connect the peptides to the Ti surface. As Figure 1, taken from that article, depicts, there is an even distribution of the PHSRN and RGD molecules; therefore, it can be seen that the co-immobilizing process was successful.

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Figure 1: Co-immobilization of PHSRN and RGD.

PHSRN-containing and RGD-containing peptides were conjugated to red and green fluorescent probes, respectively. Co-immobilization was assessed by merging the fluorescent images, which produced a yellow signal. The bioactivity of the peptide-containing surface was assessed by the adhesion, proliferation and differentiation of MC3T3-E1 murine osteoblasts.

The morphology of the MC3T3 cells, grown on various Ti surfaces, was assessed by immunofluorescence (Figure 2a-d). Cells cultured on surfaces containing the two peptides, Ti/CPTES/Mix, and cells grown on the positive-control TCPS (not shown) presented well-defined cytoskeletons and large areas with adhesion points, when compared to cells grown on untreated Ti surfaces or Ti surfaces containing only one of the two peptides. These results show that the two peptides have a synergistic effect which induces osteoblasts adhesion. The number of cells adhered to the Ti/CPTES/Mix and Ti/CPTES/RGD surfaces after two hours was significantly higher than all other surfaces, and after four hours the Ti/CPTES/Mix surface showed significantly higher results compared to all other surfaces, treated or untreated.

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Figure 2: Adhesion of MC3T3 osteoblasts. Figure 2: Adhesion of MC3T3 osteoblasts. Fluorescent imaging of MC3T3 cells cultured on (a) untreated Ti, (b) Ti/CPTES/PHSRN, (c) Ti/CPTES/RGD and (d) Ti/CPTES/Mix. Red: actin filaments (cytoskeleton); Red: vinculin (focal adhesion points); Blue: nuclei. (e) Mean ± SD of adhered MC3T3-E1 cells on the tested surfaces after 2 and 4 h in culture. Error bars represent standard deviations of at least three samples in each group. Bars with different symbols (#, O, X) are from surfaces with statistically significant differences (p-value < 0.05) at the same time point.

The proliferation of the MC3T3 cells on the various surfaces was measured after three and five days in culture. As Figure 3 shows, the Ti/CPTES/Mix and the positive control TCPS surfaces significantly enhanced the proliferation of the osteoblasts, when compared to all other surfaces.

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Figure 3: Proliferation of MC3T3 osteoblasts. Mean ± SD of proliferated MC3T3 cells on different surfaces after three and five days in culture. Error bars represent standard deviation of at least three samples in each group. Bars with different symbols (#, O, X) are from surfaces with statistically significant differences (p-value < 0.05) at the same time point.
Osteoblast differentiation was measured by alkaline phosphatase (ALP) activity (Figure 4), after seven and fourteen days of culture. The ALP levels measured for the Ti/CPTES/Mix were higher than all other titanium surfaces, indicating a high level of osteoblasts differentiation. These differences, however, were not statistically significant.

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Figure 4: Differentiation of MC3T3 osteoblasts. ALP activity (mean± SD) of MC3T3-E1 cells differentiated on different surfaces after seven and fourteen days in culture. Error bars represent standard deviations of at least three samples each groups. Bars with different symbols (#, O, X) are from surfaces with statistically significant differences (p-value < 0.05) at the same time point.

The data collected in this study shows that titanium surfaces with coatings containing the combination of motifs of two peptides, RGD and PHSRN, were bioactive and enhanced osteoblasts proliferation, adhesion and differentiation. This retained bioactivity is essential for the appropriate biological performance that is required from dental and orthopedic implants.

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