Characterization of the Protein Corona of Three Chairside Hemoderivatives on Melt Electrowritten Polycaprolactone Scaffolds

Abstract

In tissue engineering, the relationship between a biomaterial surface and the host's immune response during wound healing is crucial for tissue regeneration. Despite hemoderivative functionalization of biomaterials becoming a common tissue-engineering strategy for enhanced regeneration, the characteristics of the protein-biomaterial interface have not been fully elucidated. This study characterized the interface formed by the adsorbed proteins from various hemoderivatives with pristine and calcium phosphate (CaP)-coated polycaprolactone (PCL) melt electrowritten scaffolds. PCL scaffolds were fabricated by using melt electrospinning writing (MEW). Three hemoderivatives (pure platelet-rich plasma (P-PRP), leucocyte platelet-rich plasma (L-PRP) and injectable platelet-rich fibrin (i-PRF)) and total blood PLASMA (control) were prepared from ovine blood. Hemoderivatives were characterized via SEM/EDX, cross-linking assay, weight loss, pH and protein quantification. The interface between PCL/CaP and hemoderivative was examined via FTIR, XPS and electrophoresis. i-PRF/PCL-CaP (1653 cm^-1), PLASMA/PCL-CaP (1652 cm^-1) and i-PRF/PCL (1651 cm^-1) demonstrated a strong signal at the Amide I region. PLASMA and i-PRF presented similar N1s spectra, with most of the nitrogen involved in N-C=O bonds (≈400 eV). i-PRF resulted in higher adsorption of low molecular weight (LMW) proteins at 60 min, while PLASMA exhibited the lowest adsorption. L-PRP and P-PRP had a similar pattern of protein adsorption. The characteristics of biomaterial interfaces can be customized, thus creating a specific hemoderivative-defined layer on the PCL surface. i-PRF demonstrated a predominant adsorption of LMW proteins. Further investigation of hemoderivative functionalized biomaterials is required to identify the differential protein corona composition, and the resultant immune response and regenerative capacity.

Keywords: hemoderivative; polycaprolactone; protein corona; regeneration.

Figure 1

(A) SEM images from MEW scaffolds showing CaP surface deposition (far right image, white arrows) confirmed by energy-dispersive X-ray spectroscopy (EDX), as previously reported by our group [42]. (B) Surface characteristics of the different hemoderivatives including PLASMA.

Figure 2

Characterization of the fibrin network within the different hemoderivatives. Fibrin fiber diameter (A), pore size (B) and porosity (C) were determined via SEM images. Cross-linking (D), weight loss (E) and pH (F) using media calibrated a pH 7.35 physiological value (red dotted line) as reference. Results are expressed in mean and standard deviation. Statistically difference was determined at (**** p < 0.0001), (*** p < 0.001), (** p < 0.01) and (* p < 0.05).

Figure 3

(A) Total protein quantification for each hemoderivative. (B) At 20% dilution, no statistically significant differences were found between the hemoderivatives applied on CaP scaffolds. (C) Hemoderivative protein adsorption at different dilutions showing statistically significant starts over time with increasing dilution in P-PRP and L-PRP. Other statistical significance marked by asterisks (**** p < 0.0001), (*** p < 0.001), (** p < 0.01) and (* p < 0.05).

Figure 4

FTIR spectra for the various hemoderivatives and CaP scaffold combination. (A) All peaks in the N-H stretching region are significant above 3400 cm⁻¹, especially on PLASMA/PCL/CaP. (B) Magnification of the stretching region encompassing 1800–400 cm⁻¹ confirmed apatite deposition on PCL (1028, 600 and 561 picks) and the predominant α-Helix structural motif for i-PRF and predominant β-sheet motif for the anticoagulated protocols (i.e., P-PRP, L-PRP observed in the Amide I region). Colored bands represent Amide A (orange/N-H) or B (purple/C-H) from 3300 and 2900 cm⁻¹ respectively, as well as Amide I (blue/ C=O) which appear at lower wavelengths from 1650 cm⁻¹.

Figure 5

(A) C1s narrow scan and (B) N1s narrow scan XPS spectra for the different hemoderivatives on the PCL/CaP scaffold surface. Peak deconvolution of C1s and N1s of the different groups.

Figure 6

PC characterization. (A) SD–PAGE gel of the different groups at the 5 time points. (B) Spectrum of the protein intensity at 60 min segmented by their molecular weight (MW), showing the differential PC behavior analyzed by the area under the curve. (C) Total protein dynamic binding of different groups. (D) The protein deposition on the surface demonstrated differential protein composition by MW distribution on the PCL/CaP surface interface over time. At the early stages, predominantly high MW (>90 kDa) protein deposition was observed, followed by a secondary protein binding with medium and low MW (>30 kDa).