Solid Freeform Fabrication of Scaffolds

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One key idea in tissue engineering (TE) is based upon scaffold-guided tissue regeneration. This method involves seeding porous, biodegradable scaffolds with donor cells (e.g., cultured osteoblasts, or bone marrow stromal cells for bone TE applications) and/or growth factors (e.g., IGF and BMP for bone TE applications), then culturing and implanting the scaffolds to induce and direct the growth of new, healthy tissue. The has scaffold the 3-dimensional shape of the targeted tissue. Scaffolds, however, are often limited in practical thickness due, in part, to the difficulty in getting cells deep into interior regions of scaffolds. This problem might be eliminated if cells could be simultaneously added to the scaffolds during the scaffold synthesis process. However, scaffold fabrication processes typically involve heat or toxic chemicals that would kill living cells. Similarly, the use of chemicals and heat required for scaffold synthesis is one barrier to the realization of the incremental build-up of the advanced bioreactor which we envision. In more general terms, scaffold-based tissue engineering methodologies could benefit from a manufacturing process that could fabricate scaffolds that not only have controlled spatial gradients or distributions of cells, but that also exhibit spatial control of growth factors, and scaffold materials and microstructure. (See our Tissue Engineering Tutorial for more background on the need for spatial control).

To address these issues, we are developing a new solid freeform fabrication assembly method by which scaffolds can be incrementally built up from relatively thin, prefabricated cross-sectional layers of scaffolding (e.g., approx. 1 mm thick). These layers are stacked up to form 3-D structures by mating layers together with biodegradable or non-biodegradable  fasteners. With this assembly approach, each prefabricated section can first be seeded with cells and/or growth factors before final assembly. Then normal tissue growth across the layers, in vivo, fuses the assembly together as the scaffold degrades. Several types of tissue connectors are being investigated including miniature barbs, sutures, and screws and nuts. The steps for implementing a complete CAD/CAM assembly system, as depicted in the figure above, is described in more detail in the attached CAD/CAM assembly schematic.

To demonstrate the assembly concept, we conducted in-vivo experiments at a heterotopic site in a rabbit model. Five individual layers of porous polymer/ceramic composite material (see our Materials Research link) measuring 1 mm thick x 12 mm diameter, were individually seeded with autogenous bone marrow cells at a concentration of 1 x 108 cells/ml. The layers were then assembled with nylon sutures to create a 3-D construct. Layers of the unseeded composite were assembled as controls. The experimental and control constructs were then implanted into the rectus abdominis muscle adjacent to the inferior epigastric vessels bilaterally in six-month-old, male New Zealand White rabbits as shown below in Figures a and b. The specimens remained implanted for eight weeks. In the explanted specimens, the layers of seeded polymer/ceramic composite were not discernable by thin section histology, while discrete layers were still discernable in the control group (Figure c and d). Histomorphometry was performed using thin section histology slides with H&E and Masson's Trichrome staining. The percentage of tissue per section was measured using NIH Image. Histomorphometric analysis revealed that there was a statistically significant greater amount of bone formation in the implants seeded with cells than those implants not seeded (p<0.01).
 

a. Five (1mm thick x 12mm dia.) layers of assembled composite discs, implanted within rectus abdominus muscle, adjacent to inferior epigastric artery and vein.	b. Assembly structure shown at time of implantation in rabbit model
c. Explanted seeded group at 8 wks. d. Explanted unseeded control group at 8 wks.

With is assembly approach, different materials with different microstructures, cell types and growth factors could also be used in different sections, and prefabricated vascular constructs might be embedded and assembled into the scaffold as it is being built up as one souce for capillary sprouting. For example, the assembly idea might be extended to more complex structures such as complete bone, tendon, muscle constructs as depicted below.