Rose Bengal Crosslinking to Stabilize Collagen Sheets and Generate Modulated Collagen Laminates

Abstract

For medical application, easily accessible biomaterials with tailored properties are desirable. Collagen type I represents a biomaterial of choice for regenerative medicine and tissue engineering. Here, we present a simple method to modify the properties of collagen and to generate collagen laminates. We selected three commercially available collagen sheets with different thicknesses and densities and examined the effect of rose bengal and green light collagen crosslinking (RGX) on properties such as microstructure, swelling degree, mechanical stability, cell compatibility and drug release. The highest impact of RGX was measured for Atelocollagen, for which the swelling degree was reduced from 630% (w/w) to 520% (w/w) and thickness measured under force application increased from 0.014 mm to 0.455 mm, indicating a significant increase in mechanical stability. Microstructural analysis revealed that the sponge-like structure was replaced by a fibrous structure. While the initial burst effect during vancomycin release was not influenced by crosslinking, RGX increased cell proliferation on sheets of Atelocollagen and on Collagen Solutions. We furthermore demonstrate that RGX can be used to covalently attach different sheets to create materials with combined properties, making the modification and combination of readily available sheets with RGX an attractive approach for clinical application.

Keywords: collagen type I; drug release; mechanical stability; rose bengal and green light crosslinking; swelling degree; vancomycin.

Figure 1

Overview of methods used for collagen analysis.

Figure 2

Photographs of collagen sheets and their thickness as analyzed via height gauge. Sample size: 1 × 1 cm². (C) Collagen Solutions, (V) Viscofan and (A) Atelocollagen.

Figure 3

Swelling behavior and microstructure of collagen sheets. (A) Swelling degree of collagen sheets “as received” at 37 °C and RT after 2 h, 4 h and 24 h in relation to their dry weight (100%). (B) transmitted light microscopy (TLM) and SEM images of collagen sheets. Samples analyzed via TLM were conditioned in phosphate-buffered saline (PBS). TLM magnification: 200×. SEM magnification: V and A: 1000×, C: 200×. C: Collagen Solutions, V: Viscofan and A: Atelocollagen.

Figure 4

Impact of rose bengal and green light collagen crosslinking (RGX) on swelling degree and microstructure. (A) Swelling degree of unmodified and modified (RGX: 10 min, 0.01% rose bengal (RB)) collagen sheets after 2 h at 37 °C in relation to their dry weight (100%). (B–D) TLM and SEM images of collagen sheets before (unmodified) and after RGX (10 min, 0.01% RB). Samples analyzed via TLM were conditioned in PBS. (B) Collagen Solutions. TLM magnification: 50×. SEM magnification: 200× and 1000× (inset). (C) Viscofan. TLM magnification: 200×. SEM magnification: 1000×. (D) Atelocollagen. TLM magnification: 200×. SEM magnification: 1000×; C: Collagen Solutions, V: Viscofan and A: Atelocollagen.

Figure 5

Thickness of unmodified and modified (RGX: 10 min, 0.01% RB) collagen sheets at 37 °C and RT. Samples were conditioned in PBS for 24 h before measurement. (A) Collagen Solutions, (B) Viscofan, (C) Atelocollagen.

Figure 6

Proliferation of (A) human osteoblasts and (B) primary human muscle cells on (1) unmodified and (2) modified (RGX: 10 min, 0.01% RB) collagen sheets for 1, 7 and 10 days. The results are presented in percentage of the control without collagen sheet (100%). C: Collagen Solutions, V: Viscofan and A: Atelocollagen.

Figure 7

Release of vancomycin from unmodified and modified (RGX: 10 min, 0.01% RB) collagen samples over the course of 24 h. Collagen sheets were loaded with 1000 µg vancomycin (100%). (A) Collagen Solutions, (B) Atelocollagen.

Figure 8

Thickness of collagen laminates consisting of Collagen Solutions collagen (C) and Atelocollagen (A) prepared by RGX (10 min, 0.1% RB) compared to their theoretical thickness (sum of thicknesses of collagen single sheets) of unmodified and modified samples (RGX) at 37 °C and RT. Samples were conditioned in PBS for 24 h before measurement.