Collagen vitrigels for cornea reconstruction: collagen source and crosslinking

Abstract

 

Purpose

 

Collagen vitrigels are transparent biomaterials characterized by a highly organized, fibrillar nanostructure. We evaluated the effect of manufacturing vitrigels with collagen from bovine dermis (Bov), human fibroblast cell culture (HuCC) and recombinant human collagen expressed in tobacco leaves (rH) on the resulting membrane fibrillar organization. We further enhanced physical properties of the material through crosslinking (XL).

 

Methods

 

Vitrigels were prepared as previously described (Biomaterials 33 (2012) 8286-95) with slight variations for the different collagen types. Selected membranes were subsequently crosslinked using a standard EDC-NHS method. Transmittance was measured using a Synergy 2 microplate reader (Biotek). Differential scanning calorimetry (DSC) was used to identify the denaturation temperature of the vitrigels. Scanning was performed at a heating rate of 10°C/min using a DSC8000 machine (Perkin Elmer). Samples were processed for TEM and stained with uranyl acetate; images were taken in a Philips CM120 TEM.

 

Results

 

Collagen membrane ultrastructure was dependent on the collagen source. Fibril alignment was evident in some areas of the rH vitrigels (Figure 1), whereas Bov and HuCC showed a random fibrillar organization. Denaturation temperatures, transmittance at 550 nm and thickness data for Bov, HuCC, rH and their crosslinked counterparts (Bov XL, HuCC XL and rH XL) are shown in Figure 2. XL of the membranes increased the denaturation temperature in all cases, suggesting improved thermal stability of the collagen organization in the materials. The greatest denaturation temperature change resulting from XL was seen in rH (15.81°C), and the lowest in Bov (6.97°C). There was also a clear trend towards an increase in light transmission after XL in all groups. There was a 21.74%, 18.18% and 12.79% decrease in thickness in Bov, HuCC and rH vitrigels, respectively, after crosslinking.

 

Conclusions

 

Collagen source has a direct influence on vitrigel formation, biomaterial ultrastructure and XL effect. rH vitrigels show remarkable regional fibril alignment. Although untreated rH vitrigels are less thermally stable, they may be reinforced through XL to levels higher than Bov. rH vitrigels have the least loss of thickness after EDC-NHS treatment. Vitrigel clarity can be enhanced through XL.

 

 

Figure 1. Fibril alignment in rH collagen vitrigels (Bar=500 nm)

 

 

Figure 2. DSC, transmittance and thickness data

 

Keywords: 480 cornea: basic science