Patent Application
20120315699
Invention presented aims to improve the efficiency of biomaterials and tissue engineering scaffolds by introducing precise control of surface topography in a 3D biomaterial or tissue engineering construct by using polymeric nano and micropatterned building blocks.
As an alternative method to transplantation for remediation of tissue damage or loss, tissue engineering utilizes scaffolds which are permissive to cell growth and can be degraded and remodeled under in vivo conditions. Most tissue engineering scaffolds are designed to provide sufficient space for cells to grow in and have random porosity to allow diffusion of molecules and cells. It has been shown that remodeling process can be strongly affected if physical or chemical cues are presented to the cells on the surface of tissue engineering scaffolds. Responses of the cells range from cell orientation to aligned extracellular matrix secretion to more subtle changes such as degree of differentiation. For many tissues, intricate extracellular matrix structure is crucial for the functionality of the tissue, and tissue properties generally depend on the orientation of ECM molecules such as collagen and elastin and distribution of the cells.
Natural tissues generally contain more than one cell type in each individual layer arranged in a specific spatial orientation with respect to each other. This orientation and separation is essential for the functionality of these tissues. As an example, cornea tissue contains 5 distinct layers and 3 different cell types, the spatial organization of which should be imitated in order to produce an artificial cornea. Thus, a tissue engineering scaffold for complex tissues with more than one cell type should provide necessary separations between different cell types and at the same time should allow interaction between different cell types through physical and chemical cues available.
Most of the current tissue engineering scaffold designs either have homogeneous forms, such as foams with a random distribution of the pores, or have 2D or 3D features restricted to surface, which in turn can just affect 2D organization of the cells. Different cell types react to different range of topographies (type and magnitude) and accurate simulation of these differences on the tissue engineering scaffolds would improve their efficiency. For example, it has been demonstrated that responses of corneal epithelial and stromal cells to surface cues are distinctly different and the size of the optimal surface topographical feature for each type of cell are different. Thus, a 3D construct with lamella with unique 2D or 3D properties can provide different topographical features for each layer and allow the creation of a scaffold suitable for a complex, multilayer, multi-cell tissue. Thus biomaterials designed for tissue engineering and non-tissue engineering purposes will benefit from the construct developed.
In addition cell free biomaterials with patterned layers and stacked to form multilayer constructs may also be preferable to single layer, unpatterned biomaterials due to their increased organization and thus mimicking more closely the tissue they mimic and/or replace.
The present invention describes a 3D multilamellar construct manufactured from preproduced individual lamella, of either natural or synthetic polymer origin, which have micro- or nano-scale surface features designed to affect biomaterial performance or cell behavior. This invention includes different methodologies developed for different polymers for preparation of 3D construct, and 3D scaffolds with different dimensions and designs both physical and chemical, with respect to the orientation of lamellae, and different size surface features and multilamellar structures of different thicknesses. It also relates to the application of these structures generally to biomaterials and specifically to tissue engineering of scaffolds for tissues, and especially for a specific target tissue, cornea.
The exemplary demonstration of the present invention, is made with two different polymeric substances, 1) polyesters poly(L-lactide-co-D,L-lactide) ((P(L/DL)LA) and poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV), and 2) collagen. Briefly, collagen and P(L/DL)LA-PHBV membranes with topographical features at micro scale were produced by using photolithography and soft lithography techniques followed by solvent casting. Then solid membranes were brought together by heat application to specific contact points determined with careful consideration of the mechanical properties desired for the specific application. The second method developed is the application of an appropriate solvent in minute amounts to the contact points for local wetting followed by drying process. A third method includes the application of crosslinker solutions, in which the strength of attachment of two layers can be controlled by the concentration, amount and type of crosslinker.
The following detailed description of an embodiment of the invention and related drawings, figures and their descriptions are only of exemplary nature and thus should not be regarded restrictive or illustrative. Further features and aspects of the presented invention will become apparent to those of ordinary skill in the art upon reading and understanding the following detailed description.
The accompanying drawings, which are incorporated into and form a part of the specification, schematically illustrate one or more exemplary embodiments of the invention and, together with the general description given above and detailed description of the preferred embodiments given below, serve to explain the principles of the invention.
a. SEM micrograph of a patterned collagen film
b. Stereomicrograph of a patterned collagen film (Magnification ×30)
c. Stereomicrograph of a collagen film-based, 3D multilayer construct. In each layer the pattern direction is orthogonal to the subsequent layer. Because of the transparency of the collagen films lower layers can be seen.
d. Fluorescence micrograph (DAPI staining for cell nuclei) of a collagen film multilayer, seeded with human corneal keratocytes, after 14 days of incubation.
e. Fluorescence micrograph (DAPI staining for cell nuclei) of a single, patterned collagen film layer, seeded with another type of cell, D407 retinal pigment epithelium cells, after 7 days of incubation.
f. Fluorescence micrograph (Acridine orange staining for cell nuclei) of a single, patterned collagen film, seeded with human corneal keratocytes, after 7 days of incubation.
g. Fluorescence micrograph showing distribution of FITC-labeled phalloidin staining of cytoskeletal element (f-actin) and orientation of human corneal keratocytes on single layer of patterned collagen films after 7 days of incubation.
h. Proliferation rate of human corneal keratocytes on a 3 layered collagen film multilayer as determined by Alamar Blue assay. Tissue culture polystyrene was used as the control.
a. SEM micrograph of a bilayer of A3 patterned (P(L/DL)LA-PHBV films. Films were brought together in an orientation where the pattern axes were orthogonal to each other.
b. Fluorescence micrograph (Acridine orange staining for cell nuclei) of D407 retinal pigment epithelium cell-seeded, patterned (P(L/DL)LA-PHBV film after 1 day of incubation.
c. Fluorescence micrograph (Acridine orange staining for cell nuclei) of D407 retinal pigment epithelium cell-seeded patterned (P(L/DL)LA-PHBV film, after 7 days of incubation. Pattern dimensions are different than the others.
d. Fluorescence micrograph (DAPI staining for cell nuclei) of human corneal keratocyte seeded, patterned (P(L/DL)LA-PHBV films, after 21 days of incubation.
a Light micrograph of template (micropatterned Si template—×350) (Sm: smooth-unpatterned region, MP micropatterned region)
b Light micrograph of polymeric film obtained from the template (micropatterned PHBV-P(L/DL)LA film—×100) (Sm: smooth-unpatterned region, MP micropatterned region)
In the processes developed in this invention polymers of natural or synthetic origin were used.
Solutions of collagen and solutions of blends of natural and synthetic origin polyesters in different ratios with different concentrations were prepared.
As an example to polyesters, blends of P(L/DL)LA and PHBV were used. Solutions were poured onto patterned templates either produced on silicon wafers by photolithography or obtained by transferring the “parallel grooves and ridges” designs from primary templates made of silicon wafers onto secondary templates. Membrane structures which have inverse surface patterns of the template were produced by solvent casting. Template structure could be of any topographical feature, such as ridges or grooves connected by inclined surfaces of any inclination degree and varying ridge and groove dimensions. Any type of micropattern such as cobblestone, pillar, 2D stripes, square, circular, etc. could be obtained either by photolithography or for nano scale patterning by electron beam lithography or interference lithography or embossing or contact printing or AFM based lithography to accommodate the necessities of any 3D design.
Since the 3D structure of natural polymers is generally very sensitive to harsh treatments a mild chemical method for construction of 3D collagen multilayer was invented. Collagen solution in acetic acid was poured onto micropatterned templates and after solution was air dried, the collagen films formed were peeled off. As the collagen solution different solutions can be used. As an example 0.2 mL, 15 mg/mL in 0.5 M acetic acid can be given. To stabilize these films a crosslinking procedure was carried out. Their crosslinking was achieved by incubation in EDC and NHS. As an example crosslinking can be achieved by incubation in 33 mM EDC and 6 mM NHS in 50 mM NaH2PO4 buffer (pH 5.5) for 2 h at room temperature. Constructs were washed with Na2HPO4 buffer (pH 9.1) for 1 h and then washed successively with 1 and 2 M NaCl. Attachment of several crosslinked films to each other was achieved by addition of a dilute solvent of collagen, 0.1% acetic acid. Solvent addition causes localized dissolution to a certain extent depending on the concentration and the amount of the solvent. Subsequent air drying creates a contact between the membranes due to simultaneous dissolution and drying at the locations which come into contact with the solvent.
A second technique for attaching collagen films, involving collagen solution and a concentrated crosslinking solution, was developed. Collagen solution was applied at the desired contact points to act as a glue between the two layers, and after addition of the collagen solution a concentrated crosslinking solution consisting of EDC/NHS was added to attach the collagen in the solution to the two membranes. With this method, the strength of the contact can be finely adjusted by changing parameters such as concentrations of collagen and crosslinker solution.
P(L/DL)LA-PHBV membranes were formed by solvent casting of a solution of P(L/DL)LA and PHBV in organic solvents such as chloroform or dichloromethane to produce micropatterned membranes with pattern dimensions inverse of those of the template. Micropatterned silicon templates with different dimensions and geometries were produced by photolithography and subsequent chemical etching. The formed membranes were removed by peeling (average film thickness 42 μm) and attached to each other by heat application to 4 corners, melting the polymer films at these points. Alternatively, attachment can be made as in the case of collagen constructs, by placing a droplet of solvent at the corners which causes local dissolution of the polymer to a certain extent depending on the amount of solvent. Air drying of the structure creates a contact between two membranes due to simultaneous dissolution and drying at the points which come into contact with the solvent.
The number of adhesion/contact points, the relative orientation of the surface topographical features, size and geometry of the features, dimensions of each film layer and number of layers can be adjusted during the manufacturing process according to the specific necessities of the target tissue. If a layer of tissue with each layer having a different organization and cell is required than multilayers of different orientations can be separately prepared and then brought together to create a construct with a multilayer, multiorientation structure. If an enhanced level of interaction is necessary between the different cell types present, or if an increased permeability for transference of solutes, growth factors, bioactive agents is needed films can be rendered partially porous by addition of appropriate solute particles of desired dimensions and their subsequent dissolution by a proper solvent which only dissolves these particles and not the film material. Similar property may be achieved by pore formation upon exposure to particulates and electromagnetic radiation. If gradual provision of bioactive agents such as growth factors are needed these agents could be dissolved in the membrane.
Three types of films, 1) Patterned (P(L/DL)LA-PHBV films on Type I pattern, 2) Patterned (P(L/DL)LA-PHBV films on Type II pattern, and 3) Patterned collagen films on inverse Type I pattern, were obtained. Films were produced by solvent casting as described previously and the geometry and dimensions of the patterns are given in Table 1.
Patterned (P(L/DL)LA-PHBV films were brought together by heat treatment at the edges of the films. By this method up to 8 layers of (P(L/DL)LA-PHBV films were brought together successfully.
Patterned collagen films were brought together by application of collagen and EDC/NHS solutions successively. Up to 3 layers of collagen films were stuck to each other by this method.
Since these 3D constructs were prepared especially for cornea tissue engineering purposes, orientation of the patterns with respect to each other was perpendicular in order to mimic natural corneal stroma structure.
Multilayers were sterilized by immersing in sterile EtOH (70%) for 2 h at 4° C. Constructs were then washed 4 times with phosphate buffer saline (PBS).
Human keratocytes culturing was started at passage 2 of a primary cell line and propagated until passage 8. In all experiments keratocytes between passages 4-8 were used. The composition of the keratocyte medium for 500 mL was as follows: 225 mL of DMEM high glucose, 225 ml of Ham F12 medium, 50 mL of new born calf serum, 10 ng/mL human recombinant b-FGF, amphotericin (1 μg/mL), streptomycin (100 μg/mL) and penicillin (100 UI/mL) at 37° C., 5% CO2 in a carbon dioxide incubator. The cells were passaged using 0.05% trypsin-EDTA solution.
D407 retinal pigment epithelium cells (passage 5 to 15) were cultivated in high glucose DMEM supplemented with 5% fetal bovine serum (FBS), 100 units/mL penicillin and 100 units/mL streptomycin at 37° C., 5% CO2 in a carbon dioxide incubator. The cells were passaged using 0.05% trypsin-EDTA solution.
Keratocytes and D407 cells were detached from the tissue culture flasks by using 0.05% trypsin for 5 min at 37° C., then centrifuged for 5 min at 3000 rpm and resuspended in their respective media. Cell number was counted using NucleoCounter (ChemoMetec A/S, Denmark). 50 000 cells/20 μL were seeded on each construct and the constructs were not disturbed for 30 min to allow cell attachment. After 30 min, 500 μL of media was supplemented to each construct. They were incubated in a CO2 incubator (5% CO2, 37° C.) for 21 days. The medium was refreshed every day. Tissue culture polystyrene (TCPS) was used as the control.
For SEM, specimens were washed with cacodylate buffer (0.1 M, pH 7.4) and distilled water and freeze dried. Samples were examined with SEM after being sputter coating with gold.
For fluorescence microscopy (IX 70, Olympus, Japan), specimens were first fixed with glutaraldehyde (2.5%) for 2 h and then washed twice with phosphate buffered saline (PBS) (10 mM, pH 7.4). The samples to be stained with Acridine orange were washed with HCl (0.1 M) for 1 min and Acridine orange was added. After 15 min, Acridine orange was removed and the sample was washed with distilled water. The cells were observed under the fluorescence microscope at the excitation wavelength range of 450-480 nm.
For DAPI and Phalloidin staining, cells on the films were fixed with 4% formaldehyde for 15 min and washed twice with PBS. Then the cells treated with 1% Triton-X-100 for 5 min in order to permeabilize the cell membrane and washed again by PBS 3 times. Samples were then incubated in a blocking solution (1% BSA (bovine serum albumin) in PBS) for 30 min at room temperature and in staining solution for 1 h at 37° C. Staining solution was 1% Phalloidin and 0.1% DAPI in 0.1% BSA in PBS solution. After incubation, samples were washed with PBS and examined under fluorescence microscope at excitation wavelengths of 450-480 nm for Phalloidin and 330-385 nm for DAPI.
To determine the cell proliferation rate, Alamar Blue cell proliferation assay was performed. At time points 1, 4, 7, and 10 days for keratocyte seeded multilayers, medium was discarded and samples were washed several times with sterile PBS. Then 5%, 500 μL, Alamar Blue solution was added and samples were incubated in a CO2 incubator (5% CO2, 37° C.) for 1 h. After incubation, Alamar Blue containing media were collected and their absorbance at 570 and 600 nm were determined by a UV-Visible spectrophotometer. Percent reduction of the dye by the metabolic activity of the cells was then determined by using the absorption coefficients of the reduced and the oxidized dye. Cell number was then determined by using a calibration curve constructed using the reduction percentage of the dye in the presence of known cell numbers.
In this invention different methodologies have been developed for different polymers for the preparation of biomaterials and/or 3D scaffolds with different dimensions and designs, both physical and chemical, with respect to the orientation of lamellae, and different size surface features and multilamellar structure of different thickness.
The main steps of these methodologies are;
A second technique for attaching collagen films involving collagen solution and a concentrated crosslinking solution was developed.
A third technique for attachment of subsequent collagen film layers to each other is application of an adhesive such as fibrin glue or cyanoacrylate.
The number of adhesion/contact points, the relative orientation of the surface topographical features, size and geometry of the features, dimensions of each film layer and number of layers can be adjusted during the manufacturing process according to the specific requirements of the target tissue.
The template structure is any type of micropattern such as cobblestone, pillar, 2D stripes, square, circular.
The templates could be obtained either by photolithography or electron beam lithography or interference lithography or embossing, or contact printing or AFM based lithography to accommodate the necessities of any 3D design.
The designs on the templates could be at nano or micro level.
The constructs may be seeded with cells that are appropriate for the target tissue.
The constructs may be seeded with one or more than one cell type according to the cell population of the target tissue.
If layers of tissue, where each layer has a different organization and cell, is required then multilayers of different orientations can be separately prepared and brought together to create a multilayer, multiorientation, and multicell construct.
If an enhanced level of interaction is necessary between the different cell types present, or if an increased permeability for transference of solutes, growth factors, bioactive agents is needed then the films can be rendered partially porous by leaching off solute particles of desired dimensions contained in a proper solvent which only dissolves these particles and not the film material. Creation of pores may also be achieved through application of electromagnetic or particulate radiation
If gradual provision of bioactive agents such as growth factors are needed these agents could be dissolved in the films.
The process is applied to natural and synthetic polymers such as chitosan, NIPAM, PDMS, PCL, hyaluronic acid, chondroitin sulfate or blends of biodegradable and nondegradable polymers.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12531911 | Mar 2010 | US |
| Child | 13589645 | US |
