The effect of extracellular matrix proteins on the cellular response of HUVECS and HOBS after covalent immobilization onto titanium

Abstract

Biomimetic surface modifications are regarded as promising approach to stimulate cellular behavior at the interface of implant materials. Aim of the study was an evaluation of the cellular response of human umbilical cord cells (HUVECS) and human osteoblasts (HOBS) on titanium covalently coated with the extracellular matrix (ECM) proteins fibrinogen, collagen, laminin, and osteopontin. For the surface modification, titanium discs were first aminofunctionalized by plasma polymerization of allylamine. The ECM protein conjugation was performed using the linker molecule a, x-bis-N-hydroxysuccinimide polyethylene glycol (Di-NHS linker). For surface characterization, infrared spectroscopy and fluorescein isothiocyanate staining (FITC) were used to evaluate the presence and distribution of primary amines in the plasma polymer film. Real-time analyses of the respective protein conjugation processes were performed via surface plasmon resonance kinetic measurements. All ECM proteins were immobilized successfully. Furthermore, the biological functionality of the conjugated factors fibronectin and collagen could be proven as they led to a distinct stimulation of cell adhesion of HUVECS and HOBS when compared to the control group. The highest cell coverage of HUVECS was observed on fibronectin-modified surfaces with approximately 35% and on collagen with 33% after 24 h (PT: 9.4%). For laminin, no additional effect was observed, and for osteopontin, only a slight enhancement of cell adhesion was found. A similar, cell-stimulating tendency of fibronectin and collagen was seen as well after 3 and 7 days. Biomimetic surface modification via plasma polymerization is a powerful method for biomolecule conjugation with a high retention of biological functionality and offer promising clinical perspectives.

Surface Treatments and Functional Coatings for Biocompatibility Improvement and Bacterial Adhesion Reduction in Dental Implantology

“…Different precursors and plasma excitation methods, process parameters and reactor geometries influence the plasma polymerization process and lead to a diversity of plasma-chemical reactions and thus to different film properties [58]. The low pressure plasma processes are the most widely used: microwave (2.45 GHz) [59] or capacitively coupled radio frequency (13.56 MHz) [59][60][61][62][63][64][65] activated [60], in planar parallel electrode plates geometry [63][64][65], in steel chamber [59,[63][64][65][66] or glass tubes [62,67,68] reactors, applying a continuous [61,67] or pulsed plasma discharge [59,[63][64][65]69].…”

“…In the frame of biomimetic surface modifications, PPAAm coating, deposited on titanium was recently studied with respect to the covalent immobilization of extracellular matrix (ECM) proteins, such as fibrinogen, collagen, laminin, osteopontin [62] and pectins, which are plant-derived polysaccharides [70]. The cellular response elicited in human umbilical cord cells (HUVECS) [62] and human osteoblasts (HOBS) [62,70], was evaluated.…”

“…The cellular response elicited in human umbilical cord cells (HUVECS) [62] and human osteoblasts (HOBS) [62,70], was evaluated. Cell adhesion can be influenced selectively according to the surface modifying ECM protein.…”

“…In [62], the deposition of PPAAm occurred in a custom-made plasma reactor. The basic setup consists of a cylindrical Pyrex glass tube..…”

“…To produce reproducible results, plasma polymer films of a thickness of approximately 160 nm were used. The amino terminated surface, in this case, were crosslinked to the proteins of interest (fibronectin, collagen, osteopontin, and laminin) by means of a PBS solution of, x-bis-N-hydroxysuccinimide polyethylene glycol (Di-NHS linker), as shown in Figure 1 in [62]. After the surface activation by the Di-NHS linker, the proteins were immobilized by placing the samples with the upside on a 30-µL droplet of each solution for 1 h. After rinsing with PBS, samples were carefully dried under an air stream.…”