Solid Freeform Fabrication in Tissue Engineering

A Technology for Directly Fabricating 3D Living Tissue...

Creating living tissue in complex geometries is a challenging issue facing the tissue engineering community. Traditional tissue engineering techniques result in living tissue of simple, often 2D geometries. By harnessing the capabilities of Solid-Freeform Fabrication (SFF) – also known as Rapid Prototyping (RP) – we can create living tissue of arbitrary 3D shapes directly from computer-aided design (CAD) data.

Not only can patient-specific living implants be created directly from medical imaging data, but the complex 3D multi-tissue configurations of native structures can be more accurately reproduced by using SFF. For example, intervertibral discs have a very specific 3D overall shape and spatial distribution of multiple cell types; traditional tissue engineering techniques would have great difficulty reproducing these types of structures.

Novel Material

The "printing ink" is a cell-seeded alginate hydrogel. The alginate hydrogel is similar to that used for injection molding tissue engineering, but has been modified to be compatible with extrusion through a printing deposition tool. The material is also stiff enough to prevent material sag, hold its shape and be manipulated.

Printing Platform

To print the alginate in prescribed 3D geometries, we used a custom-built gantry robot platform. The gantry moves the syringe deposition tool (with algiante hydrogel contained within the syringe) and extruded the alginate along specific paths.

These paths were calcualted by taking a CAD model and slicing it vertically, then planning paths within each layer. This form a path planning is a standard within the SFF community. More advanced path planning is being explored to support complex 3D, multiple material prints involving embedded geometries and difficult overhanging structures.

Results

The cells seeded within the printed alginate was shown to be viable, as demonstrated in the Live/Dead assay image below. The Live/Dead assay confirms that the printing process does not have a substantial adverse affect on the cell viability. In addition to cell viability, cell function needed also be tested.

Cell function was assessed by checking for the production of extracellular matrix (i.e. the bulk material of cartilage produced by living cells). After time in culture, the printed structures demonstrated significant levels of GAG and collagen, two markers of cartilage extracellular matrix production. These markers indicated that the printed material developed into cartilage in ways similar to samples produced by alternative tissue engineering techniques.

Once cell viability and cell function were assessed using biochemical assays, the printing of 3D structures could be focused on. Using the algiante material and the gantry robot platform, various 3D geometries were pritned directly from CAD data. In the image below: 1) a crescent, 2) a first-order approximation of an intervertibral disc, and 3) an ovine mensicus directly from a CT scan. The overall shapes of the printed contructs closely resembled the intended geometries, which were directly prescribed from 3D CAD models.

Publications

Journal Articles

Cohen D. L., Malone E., Lipson H., Bonassar L., (2006) "3D direct printing of heterogeneous tissue implants", Tissue Engineering, Vol. 12, No. 5: 1325-1335

Conference Proceedings

Cohen DL, Lo WM, Tsavaris AM, Bonassar LJ, Lipson H. “Improved quality of 3D-printed tissue engineering constructs through enhanced mixing.” Transactions of the 55th Annual Meeting of the Orthopaedic Research Society; 2009 Feb 22–25; Las Vegas, NV.
Cohen DL, Lo WM, Tsavaris AM, Bonassar LJ, Lipson H. “Improved quality of 3D-printed tissue constructs through enhanced mixing of alginate hydrogels.” Proceedings of the TERMIS-North American 2008 Annual Conference & Exposition; 2008 Dec 7–10; San Diego, CA.
Ballyns J, Cohen DL, Malone E, Lipson H, Bonassar LJ. “Evaluation of geometric fidelity for anatomically shaped tissue engineered constructs.” Proceedings of the 2008 BMES Annual Fall Meeting; 2008 Oct 2–4; St. Louis, MO.
Cohen DL, Tsavaris AM, Lo WM, Bonassar LJ, Lipson H. “Improved quality of 3D-printed tissue constructs through enhanced mixing of alginate hydrogels.” Proceedings of the Nineteenth Solid Freeform Fabrication Symposium; 2008 Aug 4–6; Austin, TX.
Blackhart C, Staab T, Cohen D, Feller S. “Automation of forensics sample collection using real-time 3-D laser tracking and playback system.” Proceedings of LabAutomation 2007; 2007 Jan 28–31; Palm Springs, CA. p. 118.
Cohen DL, Ballyns J, Malone E, Potter H, Maher SA, Wright TM, Lipson H, Bonassar LJ. “Geometric fidelity of SFF-based methods for tissue engineering of anatomically shaped constructs.” Proceedings of the 2006 BMES Annual Fall Meeting; 2006 Oct 11–14; Chicago, IL. d. 1600.
Cohen DL, Malone E, Bonassar LJ, Lipson H. “Direct freeform fabrication technique for bio-manufacturing of pre-seeded, spatially heterogeneous, anatomically-shaped alginate hydrogel implants.” Proceedings of the 2005 International Mechanical Engineering Congress and Exposition; 2005 Nov 5–11; Orlando, FL. d. 81067.
Cohen DL, Lipson H, Bonassar LJ. “Freeform fabrication of living tissue constructs using alginate hydrogel.” Proceedings of the 2005 BMES Annual Fall Meeting; 2005 Sep 28–Oct 1; Baltimore, MD. d. 1267.
Cohen DL, Maher S, Rawlinson J, Lipson H, Bonassar LJ. “Direct freeform fabrication of living cell-seeded alginate hydrogel implants in anatomic shapes.” Transactions of the 51st Annual Meeting of the Orthopaedic Research Society; 2005 Feb 20–23; Washington, DC. d. 1781.
Cohen D, Bonassar LJ, Lipson H. “3-D printing of spatially heterogeneous pre-seeded living-cell implants using alginate hydrogel.” Proceedings of the 2004 BMES Annual Fall Meeting; 2004 Oct 13–16; Philadelphia, PA. d. 966.
Cohen DL, Malone E, Lipson H, Bonassar LJ. “Direct freeform fabrication of spatially heterogeneous living cell-impregnated implants.” Proceedings of the Fifteenth Solid Freeform Fabrication Symposium; 2004 Aug 2–4; Austin, TX. p. 720–31.

Patents

Lipson, H., Bonassar, L., Cohen, D., Malone, E., (2005) MODULAR FABRICATION SYSTEMS AND METHODS, pending.