Methods and apparatus for building complex 3D scaffolds and biomimetic scaffolds built therefrom
A novel method for building complex three-dimensional scaffolds for biomimetic applications such as in vitro organ growth, using a gelatin/sugar/water gel, ultraviolet radiation, and heat or enzyme is described. The method produces gelatin-sugar hydrogels demonstrating greater thermal stability, mechanical strength, and resistance to enzymatic degradation. The invention also provides a means to assemble the gelatin sugar hydrogel films into complex three-dimensional structures (scaffolds). To account for the native biochemical factors present in natural scaffolds, methods of conjugating such factors to the gelatin-sugar hydrogel are described. These scaffolds can then be applied for tissue culturing and organ growth. The present invention also describes a system and apparatus for constructing these complex three-dimensional scaffolds by taking advantage of the physical and chemical properties of the gelatin-sugar hydrogel.
1. A method for building three dimensional biomimetic scaffolds comprising the steps of:
(a) forming a plurality of irradiated layers, wherein each irradiated layer is created by providing a mixture of gelatin and water, irradiating a portion of the mixture with UV irradiation to form a cross-linked gel layer with at least one non-irradiated region, and dissolving the at least one non-irradiated region to leave an irradiated layer;
(b) stacking a plurality of said irradiated layers; and
(c) fusing said layers together to form a scaffold that exhibits user defined complexity extending in three dimensions.
2. The method of claim 1 wherein the mixture also contains a sugar.
3. The method of claim 2 , wherein the relative concentrations of gelatin, sugar and water in the mixture ranges from 2:1:2 (gelatin:sugar:water) w/w/w to 2:1:16 (gelatin:sugar:water) w/w/w.
4. The method of claim 2 wherein the concentration of sugar in the mixture is varied to modulate gel stiffness and ease of processing.
5. The method of claim 2 wherein the mixture is selectively irradiated by tracing a pattern over the mixture using a focused beam radiation source.
6. The method of claim 1 wherein the mixture is selectively irradiated using a diffuse radiation source and a stencil or mask and wherein the stencil or mask is a UV absorbing and/or free-radical neutralizing liquid that is sprayed over the mixture.
7. The method of claim 1 wherein following said irradiation, the non-irradiated material is removed from said gel layer to leave a cross-linked gel layer demonstrating a predetermined irregular shape.
8. The method of claim 7 , wherein said removal of said non-irradiated material is accomplished by dissolving said material in a heated solvent.
9. The method of claim 7 , wherein said cross-linked gel layer is held in place during the removal step by means of magnetic material exposed to an electromagnetic field.
10. The method of claim 1 comprising the further step of conjugating biochemical factors to said gel layer to stimulate cellular processes.
11. The method of claim 10 wherein the conjugating biochemical factors are selected from a group comprising growth factors, cellular debris, extracellular matrix debris, ligands specific for integrins, viral vectors, acids, bases, transcription factors, DNA, RNA, paracrine factors, endocrine factors, proteins, lipids, and carbohydrates.
12. The method of claim 1 comprising the further step of applying thermal radiation to said gel layer following irradiation to modulate the gel layer's stiffness.
13. The method of claim 1 further comprising the step of fusing a plurality of cross-linked gel layers together to form said scaffold by the application of a fusing means.
14. The method of claim 13 , wherein said fusing means is sugar glaze and heat.
15. The method of claim 13 , wherein said fusing means is an enzyme.