Biologically active collagen-based scaffolds: advances in processing and characterization

Abstract

A small number of type I collagen-glycosaminoglycan scaffolds (collagen-GAG scaffolds; CGSs) have unusual biological activity consisting primarily in inducing partial regeneration of organs in the adult mammal. Two of these are currently in use in a variety of clinical settings. CGSs appear to induce regeneration by blocking the adult healing response, following trauma, consisting of wound contraction and scar formation. Several structural determinants of biological activity have been identified, including ligands for binding of fibroblasts to the collagen surface, the mean pore size (which affects ligand density) and the degradation rate (which affects the duration of the wound contraction-blocking activity by the scaffold). Processing variables that affect these determinants include the kinetics of swelling of collagen fibres in acetic acid, freezing of the collagen-GAG suspension and cross-linking of the freeze-dried scaffold. Recent developments in the processing of CGSs include fabrication of scaffolds that are paucidisperse in pore size, scaffolds with gradients in physicochemical properties (and therefore biological activity) and scaffolds that incorporate a mineral component. Advances in the characterization of the pore structure of CGSs have been made using confocal and nonlinear optical microscopy (NLOM). The mechanical behaviour of CGSs, as well as the resistance to degradative enzymes, have been studied. Following seeding with cells (typically fibroblasts), contractile forces in the range 26-450 nN per cell are generated by the cells, leading to buckling of scaffold struts. Ongoing studies of cell-seeded CGSs with NLOM have shown an advantage over the use of confocal microscopy due to the ability of the former method to image the CGS surfaces without staining (which alters its surface ligands), reduced cell photodamage, reduced fluorophore photobleaching and the ability to image deeper inside the scaffold.

Figure 1.

Image of a fibroblast-seeded CGS acquired by NLOM. The cells (human dermal fibroblasts) are labelled using Calcein acetoxymethyl (AM) cell-tracking dye. (a) Raw intensity image acquired by the microscope sensor. (b) Estimation of cell (red–white) and CGS strut (blue) objects. The estimation is based on the detected emission spectrum at each pixel and the known emission spectra of collagen and calcein AM dye. Scale bars, (a,b) 40 μm.

Figure 2.

Imaging the differentiation of fibroblasts into myofibroblasts inside CGSs. About 8×10⁴ cells were seeded in 8 mm diameter CGSs supplied with complete Dulbecco’s modified Eagle medium (DMEM). Starting 24 h after seeding, seeded cells inside the CGS were supplied with complete DMEM containing 1.5 ng ml⁻¹ TGFβ1. Fresh medium containing TGFβ1 was applied every 2 days. Four scaffolds were imaged: (a) 0 h, (b) 1 day, (c) 2 days and (d) 4 days after starting TGFβ1 treatment. After 7 days of TGFβ1 treatment, the CGS diameter decreased by approximately 50% and expression of α-smooth muscle actin (an established marker for myofibroblast differentiation; Desmouliere et al. 2005) was upregulated three-fold compared with basal expression levels. Cells were stained using calcein AM cell tracking dye. All images have 515×386 μm field of view.