Establishment of Collagen: Hydroxyapatite/BMP-2 Mimetic Peptide Composites

Abstract

Extensive efforts were undertaken to develop suitable biomaterials for tissue engineering (TE) applications. To facilitate clinical approval processes and ensure the success of TE applications, bioinspired concepts are currently focused on. Working on bone tissue engineering, we describe in the present study a method for biofunctionalization of collagen/hydroxyapatite composites with BMP-2 mimetic peptides. This approach is expected to be fundamentally transferable to other tissue engineering fields. A modified BMP-2 mimetic peptide containing a negatively charged poly-glutamic acid residue (E7 BMP-2 peptide) was used to bind positively charged hydroxyapatite (HA) particles by electrostatic attraction. Binding efficiency was biochemically detected to be on average 85% compared to 30% of BMP-2 peptide without E7 residue. By quartz crystal microbalance (QCM) analysis, we could demonstrate the time-dependent dissociation of the BMP-2 mimetic peptides and the stable binding of the E7 BMP-2 peptides on HA-coated quartz crystals. As shown by immunofluorescence staining, alkaline phosphatase expression is similar to that detected in jaw periosteal cells (JPCs) stimulated with the whole BMP-2 protein. Mineralization potential of JPCs in the presence of BMP-2 mimetic peptides was also shown to be at least similar or significantly higher when low peptide concentrations were used, as compared to JPCs cultured in the presence of recombinant BMP-2 controls. In the following, collagen/hydroxyapatite composite materials were prepared. By proliferation analysis, we detected a decrease in cell viability with increasing HA ratios. Therefore, we chose a collagen/hydroxyapatite ratio of 1:2, similar to the natural composition of bone. The following inclusion of E7 BMP-2 peptides within the composite material resulted in significantly elevated long-term JPC proliferation under osteogenic conditions. We conclude that our advanced approach for fast and cost-effective scaffold preparation and biofunctionalization is suitable for improved and prolonged JPC proliferation. Further studies should prove the functionality of composite scaffolds in vivo.

Effects of mechanical stimuli on structure and organization of bone nanocomposites in rats with glucocorticoid-induced osteoporosis

“…It is important to study not only the loss of mineral phase of the bone tissue, but also diagnostics of collagen fibers, since combination of these components gives strength to the bone. It is obvious that not only mineral bone density characterizes its strength as a whole (Tadano and Giri 2012;Schuster et al 2020). The femoral neck was used in our investigation, because the majority of pathological fractures occur in this region.…”

“…In the recent years, the methods of X-ray diffraction have provoked a significant interest in the experimental medicine as an instrument for the investigation of the bone nanostructure. In our experiment, we used X-ray diffraction for the study of structural and mechanical properties of the mineral crystallites and collagen fibers in the samples of rats' femoral neck investigating crystalline lattices and correlation of the components (Clark and Iball 1957;Rogers and Daniels 2002;Tadano and Giri 2012;Bunaciu et al 2015;Piga et al 2013;Schuster et al 2020).…”

Advances in Growth Factor Delivery for Bone Tissue Engineering

“…Diffusion-dependent release is restricted when the scaffold pores are smaller than the hydrodynamic radius of the incorporated protein [ 129 ]. Control over the release rate can be possible by modifying the material degradation rate and mechanism [ 130 , 131 , 132 ]. Increasing the electrostatic attraction between GFs, such as BMP-2 and TGF-β, and the scaffold matrix can also improve the loading efficiency [ 122 ].…”

Angiogenic Potential of VEGF Mimetic Peptides for the Biofunctionalization of Collagen/Hydroxyapatite Composites

“…Collagen/hydroxyapatite composites were prepared according to a previously established protocol [33]. Briefly, the required amount of hydroxyapatite nanoparticles (10 µm powder, Fluidinova, Moreira da Maia, Portugal) was weighted, washed with TBS (tris buffered saline, Biolegend), and then equilibrated in TBS overnight.…”

“…In a previous work, we used a modified BMP-2 mimetic peptide, containing a polyglutamic acid residue (E7-tag), for the incorporation into a collagen/HA composite. The included E7-tag confers accumulated negative charges and thereby a high binding affinity to positively charged hydroxyapatite surfaces [33]. This method for immobilization has the big advantage that it needs no chemical reactions or organic solvents for cross-linking, which can influence biomolecule activity and/or physicochemical integrity of the biomaterial surface of scaffolds [20].…”