Production of highly aligned collagen lamellae by combining shear force and thin film confinement

Abstract

Load-bearing tissues owe their mechanical strength to their highly-anisotropic collagenous structure. To date, attempts to engineer mechanically strong connective tissue have failed mainly due to the lack of the ability to reproduce native collagen organization in constructs synthesized by cultured cells in vitro. The ability to influence the direction of the self-assembling collagen molecules and produce highly anisotropic structures has applications ranging from de novo engineering of complex tissues to the production of organized scaffolds for cell culture contact guidance. In this investigation we have used the simple technique of spin coating to produce highly-aligned arrays of collagen fibrils. By a simple modification of the method we have also successfully produced orthogonal collagen lamellae. Alternating collagen lamellae are frequently seen in load-bearing tissues such as cornea, annulus fibrosus, and cortical bone. Culturing of corneal fibroblasts onto aligned collagen shows that the cells adopt the organization of the fibrils. In this investigation, we observed the reversal of fibrillar growth direction or “hook” formation similar to those seen previously in a microfluidic shear-flow chamber. Although the results of this investigation clearly show that it is possible to produce small areas (O) 1 cm2 of collagen fibrils with enough alignment to guide fibroblasts, there is evidence that thin film instabilities are likely to be a significant barrier to producing organized collagen fibrils over larger areas. Successful application of this method to produce highly-controlled and organized collagenous structures will require the development of techniques to control thin film instability and will be the subject of the future work.

Functional fabrication of recombinant human collagen–phosphorylcholine hydrogels for regenerative medicine applications

“…The driving force behind the formation of the lamellar structures is unclear. The most plausible explanation has been put forward by Ruberti's group [18,30]. At the high collagen concentrations used to fabricate implants and under the confined environment of the moulds used, molecular crowding as described by Ruberti et al is present.…”

The modulation of endothelial cell morphology, function, and survival using anisotropic nanofibrillar collagen scaffolds

“…Moreover, collagen I scaffolds are biodegradable and low in antigenicity, making them suitable for transplantation. Current laboratory methods aimed at achieving orientational anisotropy in reconstituted collagen matrices include mechanical loading [38], microfluidic alignment [39, 40], flow and magnetic field induced alignment [41], electrochemical processing [42], interstitial flow [43], high magnetic field [44, 45], oriented electrospinning [46], Langmuir-Blodgett deposition [47], extrusion processes [48, 49], and spin-coating [50]. Aligned collagen I matrices produced by these methods provided for essential cell-matrix interaction and successfully guided fibroblast [51-53] and endothelial cell alignment [8].…”

Anisotropic microfibrous scaffolds enhance the organization and function of cardiomyocytes derived from induced pluripotent stem cells

“…Current approaches to fabricate aligned scaffolds include mechanical loading, 4 microfluidic alignment, 5, 6 flow and magnetic field induced alignment, 7 electrochemical processing, 8 interstitial flow, 9 high magnetic field, 10, 11 oriented electrospinning, 12 melt electrospinning, 13 Langmuir-Blodgett deposition, 14 extrusion, 15, 16 and spin-coating. 17 The aligned scaffolds produced by these methods provided for essential interactions between cells and extracellular matrix (ECM) that successfully guided cardiomyocyte alignment. 18 …”