Cellular Response to Bone Morphogenetic Proteins-2 and -7 Covalently Bound to Photocrosslinked Heparin-Diazoresin Multilayer

Abstract

Despite the plethora of research that exists on recombinant human bone morphogenetic protein-2 and -7 (rhBMP-2 and rhBMP-7) and has been clinically approved, there is still a need to gain information that would allow for their more rational use in bone implantology. The clinical application of supra-physiological dosages of these superactive molecules causes many serious adverse effects. At the cellular level, they play a role in osteogenesis and cellular adhesion, migration, and proliferation around the implant. Therefore, in this work, we investigated the role of the covalent binding of rhBMP-2 and rhBMP-7 separately and in combination with ultrathin multilayers composed of heparin and diazoresin in stem cells. In the first step, we optimized the protein deposition conditions via quartz crystal microbalance (QCM). Then, atomic force microscopy (AFM) and enzyme-linked immunosorbent assay (ELISA) were used to analyze protein-substrate interactions. The effect of the protein binding on the initial cell adhesion, migration, and short-term expression of osteogenesis markers was tested. In the presence of both proteins, cell flattening and adhesion became more prominent, resulting in limited motility. However, the early osteogenic marker expression significantly increased compared to the single protein systems. The presence of single proteins resulted in the elongation of cells, which promoted their migration activity.

Keywords: bone morphogenetic protein-2; bone morphogenetic protein-7; cell culture surfaces; diazoresin; heparin.

Figure 1

Schematic presentation of the preparation of different substrates.

Figure 2

(a) rhBMP-2, (b) rhBMP-7, and (c) rhBMP-2/rhBMP-7 adsorption/desorption run for the Au/SiO₂ sensor coated with non-photocrosslinked (DR/HP)₆, expressed as the mass shift (red curves) and dissipation shift (black curves). rhBMPs were deposited from 1 μg/mL solution in 1.0 × 10⁻² M NaCl at pH 6.2 and a flow rate of 0.15 mL/min. rhBMPs were deposited for about 30–60 min (marked as light red or light blue areas) and points A and B represent the adsorbed protein mass.

Figure 3

Representative topography images of (DR/HP)₆ multilayers immersed in 1 × 10⁻² M NaCl before and after photocrosslinking and after the deposition of rhBMP-2 and rhBMP-7 from 1.0 µg/mL solutions. Measurements were performed at room temperature using the tapping mode and were collected from two areas. The corresponding root mean square roughness (R_q) values are shown in each image.

Figure 4

(a) Cell viability evaluated after 24, 48, and 72 h of hUC-MSC culture on (DR/HP)₆—control, (DR/HP)₆_BMP-2, (DR/HP)₆_BMP-7, and (DR/HP)₆_BMP-2/-7. (b) Proliferation evaluated after 2, 3, and 7 days of hUC-MSC culture on (DR/HP)₆—control, (DR/HP)₆_BMP-2, (DR/HP)₆_BMP-7, and (DR/HP)₆_BMP-2/-7. The results with p values less than 0.05 (p < 0.05) were considered to be statistically significant in comparison to the control and were labeled with an asterisk (*).

Figure 5

Actin cytoskeleton architecture of hUC-MSCs. Cells were cultured for 24 h on (rows from top to bottom) (DR/HP)₆ polymeric system without proteins (control) and with immobilized rhBMP-2 or rhBMP-7 and both proteins combined, in a medium supplemented with 2% FBS. Cells were immunostained with vinculin and counterstained with TRITC-phalloidin (F-actin) and DAPI (nuclei). The scale bar represents 25 µm.

Figure 6

(a) Cell area 24 h after seeding measured for 20 cells cultured on various surfaces. Results with p values less than 0.05 (p < 0.05) were considered to be significantly different from the control and were labeled with an asterisk (*). (b) hUC-MSC migration into the “wound”. Cells cultured on (DR/HP)₆, (DR/HP)₆_BMP-2, (DR/HP)₆_BMP-7, and (DR/HP)₆_BMP-2/-7 were monitored at the time points of 0, 2, 4, 8, and 12 h. (c) Micrographs of hUC-MSC migrating into the scratched cell-free areas taken immediately after “the injury” and after 12 h of continuous cell migration into the wound.

Figure 7

The expression of selected osteogenic gene markers in hUC-MSC cultured for 7 days on (DR/HP)₆—control, (DR/HP)₆_BMP-2, (DR/HP)₆_BMP-7, and (DR/HP)₆_BMP-2/-7. Quantitative real-time PCR analysis of gene expression of ALPL—alkaline phosphatase; RunX2—Runt-related transcription factor 2; and OCN—osteocalcin. Gene expression was normalized to the expression of the housekeeping gene GAPDH—glyceraldehyde-3-phosphate dehydrogenase—and shown as the fold change compared to the gene expression of hUC-MSC cultured on the (DR/HP)₆ surface (blue line). Results with p values less than 0.05 (p < 0.05) were considered to be significantly different in comparison to the control and are labeled with an asterisk (*).