Biosynthetic PCL-graft-Collagen Bulk Material for Tissue Engineering Applications

Abstract

Biosynthetic materials have emerged as one of the most exciting and productive fields in polymer chemistry due to their widespread adoption and potential applications in tissue engineering (TE) research. In this work, we report the synthesis of a poly(ε-caprolactone)-graft-collagen (PCL-g-Coll) copolymer. We combine its good mechanical and biodegradable PCL properties with the great biological properties of type I collagen as a functional material for TE. PCL, previously dissolved in dimethylformamide/dichloromethane mixture, and reacted with collagen using carbodiimide coupling chemistry. The synthesised material was characterised physically, chemically and biologically, using pure PCL and PCL/Coll blend samples as control. Infrared spectroscopy evidenced the presence of amide I and II peaks for the conjugated material. Similarly, XPS evidenced the presence of C-N and N-C=O bonds (8.96 ± 2.02% and 8.52 ± 0.63%; respectively) for PCL-g-Coll. Static contact angles showed a slight decrease in the conjugated sample. However, good biocompatibility and metabolic activity was obtained on PCL-g-Coll films compared to PCL and blend controls. After 3 days of culture, fibroblasts exhibited a spindle-like morphology, spreading homogeneously along the PCL-g-Coll film surface. We have engineered a functional biosynthetic polymer that can be processed by electrospinning.

Keywords: biosynthetic; collagen; conjugation; electrospinning; poly(ε-caprolactone); tissue engineering.

Figure 1

Schematic representation of the Poly-ɛ-caprolactone-graft-Collagen (PCL-g-Coll) material preparation. (A) Step-by-step preparation process of formulated biosynthetic material; and (B) Schematic representation of PCL and collagen coupling reaction by carbodiimide chemistry.

Figure 2

(A) Fourier Transform Infrared Spectroscopy in Attenuated Total Reflection Mode (FTIR/ATR) of PCL, Collagen, PCL/Coll blend, PCL-g-Coll samples; (B) The typical band of Amide I and II in order to detect the presence of collagen.

Figure 3

(A) XPS Survey; (B) XPS high resolution of PCL, PCL/Col blend and PCL-g-Coll samples.

Figure 4

(A) Contact angle and (B) Mechanical properties (Tensile test and DMS) of PCL, PCL/Coll blend, PCL-g-Coll (* p < 0.05).

Figure 5

Collagen staining of (A) PCL; (B) PCL/Coll blend and (C) PCL-g-Coll with Sirius Red assay. Scale bars correspond to 400 μm.

Figure 6

Sample weight after in vitro degradation tests in PBS at 37 °C (n = 3, * p < 0.05 and ** p < 0.001). The values of each type of sample are normalised against their corresponding initial weight.

Figure 7

L929 cell response to PCL, PCL/Coll blend, PCL-g-Coll: (A) Live/Dead assay (scale bar = 100 μm); (B) Cellular staining with rhodamine phalloidin for the actin filaments (red), focal adhesion protein vinculin (green) and nuclei counterstained with DAPI (blue).

Figure 8

Metabolic activity of L929 cell response to PCL, PCL/Coll blend, and PCL-g-Coll (** p < 0.001) measured by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. The values are normalised against tissue culture plastic control.

Figure 9

(A) Scanning Electron Microscopy (SEM) micrographs of the electrospun membranes composed by PCL (above) and PCL-g-Coll (below); (B) Zoomed-in image of the significant wavelength band of the FTIR/ATR for PCL and PCL-g-Coll electrospun membranes.