Three-dimensional (3D) tissue scaffold with cell alignment

A method of preparing a 3-dimensional (3D) construct that supports cell attachment and alignment across three dimensions, comprising the steps of:

• a) providing a biocompatible 2D laser-ablatable polymer substrate, optionally in sheet form;
• b) coating said polymer substrate with a first photoresist to produce a photoresist-protected polymer substrate;
• c) forming a pattern of single holes through said photoresist-protected substrate to produce a perforated protected substrate,
• wherein each of the elongated holes has a length dimension up to about 130 μm and a width dimension of about 20 to 40 μm, and
• wherein the elongated holes are arranged in rows such that holes in one row are offset from holes in a neighboring row so that holes in one row are staggered with those in the neighboring row in a direction of row progression;
• d) removing the first photoresist from said perforated protected substrate to produce a perforated polymer substrate;
• e) coating said perforated polymer substrate with a second photoresist coating pattern to pattern the perforated polymer substrate with a parallel-stripe, exposed polymer surface, wherein the pattern of the photoresist-covered perforations extends along the exposed polymer substrate in an orientation parallel to the stripe, exposed polymer surface;
• f) forming a coating of a cell-adhesive organometallic/phosphonate adduct on said parallel stripes of exposed polymer substrate on the surface of said perforated polymer substrate, wherein said organometallic/phosphonate adduct comprises an organometallic layer covered with a phosphonate layer, and wherein the phosphonate layer comprises a self-assembled monolayer of a cell-adhesive phosphonate (SAMP) ligand;
• g) removing said photoresist from said perforated polymer substrate to form a 2D construct coated with organometallic/phosphonate adduct stripes in micron-dimensioned continuous parallel lines, wherein said construct supports cell attachment and alignment across two dimensions;
• and
• h) stacking multiple said 2D constructs that support cell alignment and attachment across two dimensions to form a 3D construct,
• wherein said micron-dimensioned parallel stripe-patterned layer is dimensioned to (1) template attachment and proliferation of cells in register with the stripe pattern so that cell proliferation is aligned in the linear stripe direction and remains aligned over the entire surface and (2) allow the cells to grow to confluence, and
• wherein on each 2D construct, every cell is no more than about 200 μm from at least one elongated hole.

A 3-dimensional (3D) construct that supports cell attachment and alignment along cell-adhesive continuous parallel stripe patterns, comprising stacked layers of a coated 2-dimensional (2D) construct that supports cell attachment and alignment across two dimensions, each 2D construct comprising:

• a) a biocompatible laser-ablatable polymer substrate wherein the polymer surface is coated with a ZrO2 interface that is directly reactive with a cell-adhesive phosphonate or phosphonic acid, said substrate having a pattern of elongated holes therethrough extending across the two-dimensional surface of the substrate, wherein each of the elongated holes has a length dimension up to about 130 μm and a width dimension of about 20 to 40 μm, and wherein the elongated holes are arranged in rows such that holes in one row are offset from holes in a neighboring row so that holes in one row are staggered with those in the neighboring row in a direction of row progression;
• and
• b) a patterned layer of an α,ω-diphosphonate or α,ω-diphosphonic acid directly attached to the surface of said substrate in a self-assembled monolayer of cell-adhesive phosphonate (SAMP) ligand in micron-dimensioned continuous parallel stripes, wherein the pattern of the holes extend along the parallel cell-adhesive stripes;
• wherein the polymer substrate comprises polyacrylates,
• wherein said micron-dimensioned parallel stripe pattern is dimensioned to (1) template attachment and proliferation of cells in register with the stripe pattern so that cell proliferation is aligned in the linear stripe direction and remains aligned over the entire surface and (2) allow the cells to grow to confluence, and
• wherein on each 2D construct, every cell is no more than about 200 μm from at least one elongated hole;
• and
• further comprising a confluent cell layer attached to the coated surface of said polymer substrate,
• wherein the cells are spread around and between the holes, but do not block the holes, and are in alignment with the parallel cell-adhesive stripes.

A method of preparing a 3-dimensional (3D) construct that supports cell attachment and alignment across three dimensions, comprising the steps of:

• a) providing a biocompatible 2D laser-ablatable polymer substrate that is coated with a ZrO2 interface that is directly reactive with a cell-adhesive phosphonate, optionally in sheet form;
• b) coating said polymer substrate with a first photoresist to produce a photoresist-protected polymer substrate;
• c) forming a pattern of holes through said photoresist-protected substrate to produce a perforated protected substrate, wherein the holes are arranged in rows such that holes in one row are offset from holes in a neighboring row so that holes in one row are staggered with those in the neighboring row in a direction of row progression;
• d) removing the first photoresist from said perforated protected substrate to produce a perforated polymer substrate;
• e) coating said perforated polymer substrate with a second photoresist to pattern the perforated polymer substrate with a parallel-striped, exposed polymer surface, wherein the pattern of photoresist-covered perforations extends along the exposed polymer substrate in continuous lines in an orientation parallel to the striped, exposed polymer surface;
• f) forming a coating of a cell-adhesive α,ω-diphosphonate or α,ω-diphosphonic acid directly on the parallel stripe-coated surface of said perforated polymer substrate;
• g) removing said photoresist from said perforated polymer substrate to form a 2D construct coated with cell-adhesive α,ω-diphosphonate or α,ω-diphosphonic acid stripes directly attached to said polymer substrate in micron-dimensioned continuous parallel lines, wherein said construct supports cell attachment and alignment across two dimensions;
• and
• h) stacking multiple said 2D constructs that support cell alignment and attachment across two dimensions to form a 3D construct;
• wherein the polymer substrate is selected from the group consisting of polyacrylates, polyamides and polyurethanes, and
• wherein said micron-dimensioned parallel stripe pattern is dimensioned to (1) template attachment and proliferation of cells in register with the stripe pattern so that cell proliferation is aligned in the linear stripe direction and remains aligned over the entire surface and (2) allow the cells to grow to confluence.