Theoretical and experimental analysis of a biomimetic printer of anisotropic arrays of type I collagen fibrils or 'nanoloom'

Clinically viable approaches to therapeutic connective tissue replacement are dominated by the use of all-synthetic implants (comprising second generation biomaterials) with relatively few examples of tissue-engineered implants (e.g. artificial skin - a third generation biomaterial). A biological printer capable of depositing aligned collagen fibrils for use as implantable load bearing third generation materials is described herein. The success of artificial skin, which typically is produced by seeding fibroblastic cells into a disorganized collagen scaffold, has not translated well to applications where constructs are exposed to significant mechanical loads. To replace load-bearing tissue (such as tendon, annulus fibrosus, ligament and cornea), it is necessary to replace the highly-organized, anisotropic arrays of collagen fibrils responsible for their robust mechanical properties. Development of methods which facilitate the de novo production of organized collagenous arrays may ultimately enable the replacement of damaged load-bearing connective tissue. We here describe the design of a mechano-chemical device mimetic of the process by which fibroblasts putatively synthesize anisotropic collagen arrays (within a thin "pore" or "fibripositor"). The device, referred to as a "nanoloom", is built around a nano-porous track-etched membrane (with analogous thin "pores") and features energy, mass and ion transfer control to direct collagen self-assembly of fibrils. Physicochemical conditions designed to facilitate alignment and assembly of the collagen fibrils are produced inside 80-nm diameter nano-channels. Each nano-channel represents a single fibripositor or "nanoreactor" and millions of these nano-channels are in parallel in the membrane. The membrane is integrated into a complex, thermally-controlled printer head which is mounted to a stable, 3-degree of freedom, high-resolution gantry for accurate placement of printed fibrils. Strict thermal control over the thin membrane through the use of an optical coupling system is theoretically analyzed. Membranes are sputter coated with germanium films to absorb a high-frequency laser output to generate localized heating. Initial results without this strict thermal control system confirm the adequacy of the design. First level testing of the nanoloom printed fibril-like structures that appear to be collagen fibrils but have yet to be completely verified.