WO2013071062A1

WO2013071062A1 - Multilayered collagen tubular scaffold

Info

Publication number: WO2013071062A1
Application number: PCT/US2012/064388
Authority: WO
Inventors: Kyle ALBERTI; Qiaobing Xu
Original assignee: Tufts University
Current assignee: Tufts University
Priority date: 2011-11-09
Filing date: 2012-11-09
Publication date: 2013-05-16
Anticipated expiration: 2014-05-09

Abstract

A collagen scaffold comprising 2-10 stacked collagen fiber layers, wherein at least 90% of the collagen fibers in each layer have a length of at least 2 mm and are so oriented that they extend along the layer and are parallel to each other, each layer has a thickness of 10-100 µm and features a direction identical to the orientation of the at least 90% of the collagen fibers, and the angle between the directions of every collage fiber layers next to each other is 30°-90°. Also disclosed is a collagen tube made of the above-described collagen scaffold.

Description

MULTILAYERED COLLAGEN TUBULAR SCAFFOLD

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 61/557,660, filed November 9, 2011, the contents of which are hereby incorporated in their entirety. BACKGROUND OF THE INVENTION

Tissue engineering improves the health and quality of life by restoring, maintaining, or enhancing tissue/organ functions. For example, tissue-engineering blood vessels have been used to replace or bypass blood vessels that have narrowed or are even blocked due to disease processes.

Requirements for tissue engineering are high. Take tissue-engineered blood vessels for example. They should adapt to remodeling according to the needs of the environment. Also, they should induce little or no immunologic reaction. In addition, they should have high and long-term patency rates, high burst strength, low compliance mismatch, high infection resistance, and off-the-shelf availability. Further, they should have adequate mechanical strength so as to securely affix themselves to target sites.

Finally, not only should they be able to contract in response to hemodynamic forces and chemical stimuli, they should also secrete normal blood vessel products.

There is a need for new tissue engineering materials that meet all mechanical, biological, and functional requirements.

SUMMARY OF THE INVENTION

One aspect of this invention relates to a collagen scaffold including 2-10 stacked collagen fiber layers each having a thickness of 10-100 μιη. In each layer, at least 90% of the collagen fibers have a length of 2 mm or greater and are so oriented that they extend along the layer and are parallel to each other. Each layer features a direction identical to and defined by the orientation of the at least 90% of the collagen fibers, the direction being 30°-90° (e.g., 45°-90°, 60°-90°, and 80°-90°) relative to the direction of each collagen fiber layer next thereto. The collagen fiber layers in the collagen scaffold can be obtained from natural sources, e.g., tendon, bone, skin, and ligament.

The collagen scaffold of this invention can include 1-9 non-collagen elastic layers, each of which is deposited in between two collagen fiber layers. The collagen scaffold can also include on its surface a polymer coating, which optionally contains a biologically active agent.

Another aspect of this invention relates to a method of producing the collagen scaffold described above. The method includes first providing layers (each being 10- 100 μιη in thickness) of collagen fibers, at least 90% of which in each layer have a length of 2 mm or greater and are so oriented that they extend along the layer and are parallel to each other, thereby defining the direction of that layer; and then stacking the layers of collagen fibers in such a manner that the angle between the directions of any two layers next to each other is 30°-90°.

Still another aspect of this invention relates to a collagen tube including a tubular wall, which has the same features as those of the above-described collagen scaffold. Namely, the tubular wall includes 2-10 stacked collagen fiber layers, wherein at least 90% of the collagen fibers have a length of 2 mm or greater and are so oriented that they extend along the layer and are parallel to each other, and each layer has a thickness of 10- 100 μιη and a direction identical to the orientation of the at least 90% of the collagen fibers, the direction being 30°-90° (e.g., 45°-90°, 60°-90°, and 80°-90°) relative to the direction of each collagen fiber layer next thereto.

A further aspect of this invention relates to a method of producing a collagen tube. The method includes the steps of making the collagen scaffold as described above and further wrapping the collagen scaffold around a cylindrical object having a diameter of 0.1-10 mm and removing the cylindrical object to provide a tube having collagen tubular wall.

Also within the scope of this invention is use of the above-described collagen tube in treating vascular diseases or renal failure, in repairing nerve, or in urethral replacement.

The details of one or more embodiments of the invention are set forth in the description and the drawings below. Other features, objects, and advantages of the invention will be apparent from the detailed description of several embodiments and also from the appending claims. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a bi-layered collagen scaffold of this invention.

Figure 2 is an illustration of a process of making the multi-layered collagen scaffold of this invention.

Figure 3 is a SEM image of the collagen fiber layers of the collagen tube of this invention.

Figure 4 is an optical image of two collagen fiber layers of the collage tube of this invention.

Figure 5 is a diagram showing the stress-strain behaviors of various collagen scaffolds.

Figure 6 is an image of a part of a tube wall in which PC 12- neuron cells grow along the collagen fibers.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to collagen scaffolds including 2-10 stacked collagen fiber layers. Figure 1 shows a collagen scaffold having 2 stacked collagen layers, i.e., layers 101 and 102, each of which has a thickness (t) of 10-100 μιη. Collagen fibers (not shown) having a length of at least 2 mm extend along the layers. In layer 101, a substantial amount (e.g., at least 80%, 85%, 90%, 95%, or 99%) of the collagen fibers are parallel to each other and the orientation of these fibers, identical to the direction of the layer, is shown as direction 103. Similarly, in layer 102, the orientation of a substantial amount of the collagen fibers is shown as direction 104. The angle between the directions of layers 101 and 102 is 30°-90°, 45°-90°, 60°-90°, or 80°-90°.

For those collagen scaffolds of this invention that have 3-10 collagen fiber layers, every two collagen fiber layers next to each other are stacked in the same manner as the two collagen fiber layers shown in Figure 1. Collagen fiber layers next to each other refer to two closest collagen fiber layers that are either in contact with each other or separated by one or more non-collagen layers.

The collagen scaffolds of this invention can be prepared by the process illustrated in the scheme shown in Figure 2. Natural collagen sources are cut into small pieces and decellularized to obtain small collagen blocks. See J. Physiol. 567.3 (2005) pp 1021— 1033. The order of the cutting and decellularization steps can alter. Natural collagen sources include, but are not limited to, human or animal tendon, bone, skin, cartilage, ligament, and blood vessels. Cutting can be performed by any conventional method. The small pieces after cutting typically have a size in the range of 1-100 mm.

Decellularization can be accomplished using one or more decellularization agents selected from detergents, emulsification agents, proteases, and ionic strength solutions. See U.S. Patent 6,962,814 for suitable decellularization agents and conditions.

Decellularization preferably does not cause gross alteration in the structure of the tissue or cause substantial alteration in its biomechanical properties. The effects of

decellularization on structure can be evaluated by light microscopy and/or ultrastructural examination.

Preferably, following removal from the solution used in the decellularization, the decellularized tissue is washed in a physiologically appropriate solution, e.g., PBS or tissue culture medium. Washing removes residual decellularization solution that might otherwise cause deterioration of the decellularized tissue, inhibit the growth of subsequently seeded cells, and reduce biocompatibility.

The collagen blocks are then cut into slices having a predetermined thickness of

10-100 μιη by cryostat microtome. See also J. Physiol. 567.3 (2005) pp 1021-1033. Briefly, the collagen blocks are frozen at a low temperature and then fixed to the cutting base plate of a cryostat with optimal cutting temperature (OCT) compound, e.g., Tissue- Tekl^® (Sakura Finetek USA, Inc., Torrance, CA), which contains polyvinyl alcohol and polyethylene glycol. The frozen collagen blocks are then cut by a microtome into collagen slices. Preferably, they are cut along the direction parallel to the orientation of the collagen fibers in the blocks so that the collagen fibers extend along the obtained slices.

After collagen slices are obtained, 2-10 of them are stacked together in such a manner that the collagen fiber orientation of each collagen slice is at a desired angle between 30°-90° relative to the collagen fiber orientation in any collagen slice next to it. To form an integral collagen scaffold of this invention, the stack of collagen slices can be compressed or collagen slices can be held together by applying a binder, e.g., fibrinin glue, poly(lactic-co-glycolic acid), or silicone.

If desired, the layers (i.e., slices) of the collagen scaffold can be chemically bonded to further enhance their interaction. The collagen fibers on the surface of two adjacent layers can be cross-linked by a chemical cross-linking agent (e.g., an aldehyde compound or a carbodimiide) or heat (dehydrothermal cross-linking). Formaldehyde is a preferred cross-linking agent, given its high vapor pressure and its safe use in implantable devices. Alternatively, the collagen fibers can also be cross-linked by plasma or any other known method.

When a cross-linking agent is used, the extent of the cross-linking can be controlled by the concentration of the cross-linking agent, the time for the cross-linking reaction, and the temperature (if the agent is in liquid form), or the vapor pressure (if the agent is in gas form), under which the cross-linking reaction is carried out.

The collagen scaffolds of this invention have good isotropic and mechanical properties due to different collagen fiber orientations of neighboring layers in the scaffolds. One can control the collagen fiber orientations of the layers to obtain preferable isotropic and mechanical properties. For example, the angle of the carbon fiber orientations of two neighboring collagen fiber layers can be set to 45°-90°, 60°-90°, or 80°-90°.

The collagen scaffolds of this invention can be used as bone, tendon, skin, cartilage, and blood vessel implants. To promote tissue engineering, cells (e.g., endothelial cells or smooth muscle cells) can be seeded in the collagen scaffolds prior to implantation into a subject. In addition, the collagen scaffolds can be treated with one or more biologically active agents. The agent(s) is selected to enhance the properties of a scaffold following implantation, e.g., to facilitate populating endogenous cells (i.e., cells present within the subject) in the scaffold, to enhance the growth of seeded cells, to facilitate vascularization of the scaffold, and to reduce the likelihood of thrombus formation. Appropriate biologically active agents include, but are not limited to, thrombomodulators, hemocompatible agents, and antibiotics. In certain cases, an agent is a pharmaceutical composition intended for treating the condition the same as, or different from, that being treated by implanting the scaffold.

The collagen scaffolds of this invention can be configured to assume a particular desired shape and size by a known method. For example, a tubular collagen scaffold can be made by wrapping a collagen scaffold (prepared by the process described above) that is in sheet form around a cylindrical object having a predetermined diameter (e.g., 0.1 to 1 mm). Such a tubular collagen scaffold is suitable for use as a replacement for a damaged blood vessel or as a nerve conduit that promotes nerve growth.

Thus, one aspect of this invention relates to a collagen tube including a tubular wall, i.e., a tubular collagen scaffold, which contains 2-10 stacked collagen fiber layers. If desired, one or more biologically active agents can be incorporated into the layers of the collagen tube of this invention to promote tissue engineering of a blood vessel or nerve or to prevent tissue adhesion. Examples include but are not limited to growth factors (e.g., the vascular endothelial growth factor), cytokines, laminins,

glycosaminoglycans, glycoproteins, fibronectins, and drugs (e.g., immunosuppressive drugs). The biologically active agent can be incorporated into the layers via electrostatic interaction, physical or mechanical interaction, covalent bonding using crosslinking agents or light, a combination of the above, or via a spacer molecule that is well known in the art. For example, one can immerse collagen layers into a solution containing a biologically active agent before stacking them or immerse the stacked collagen layers into such a solution. Alternatively, one can spray a solution containing one or more biologically active agents onto a collagen scaffold or collagen tube.

The collagen tube can be coated with poly(dimethylsiloxane), poly(lactic-co- glycolic acid), polystyrene, silk, or hydrogels to improve mechanical properties of the tube. The above-mentioned biologically active agents can be included in the polymer coatings. Controlled release of these biologically active agents can therefore be achieved.

The surfaces of the collagen tube can be treated to introduce the desirable hydrophilic nature so as to support biocompatibility. The pH value of the collagen tube surfaces can also be adjusted to prevent significant systemic or local reaction. See, e.g., Bostman et al, Biomaterials 2000, 21 :2615-2621.

To improve the tensile strength of the collagen tube, one can also insert an elastic non-collagen layer between two collagen layers. "Non-collagen layer" refers to a layer containing no collagen at all or an unsubstantial amount of collagen. The elastic non- collagen layer can be made a biocompatible material, such as a natural material (e.g., a slice of an aorta vessel wall) or a synthetic material (e.g., a synthetic polymer slice). Examples of suitable synthetic polymers include poly(glycerol sebacatej, silicone, or polyurethane. These elastic layers can be introduced during the stacking step.

To sustain mechanical stress, the collagen tube may also include one or more supportive layers. Such supportive layers can be placed between any two collagen fiber layers or attached to the inner or outer surface of the collagen tube wall.

The collagen tube can further include cells (e.g., endothelial cells and smooth muscles cells) to promote tissue engineering. Typically, this process involves seeding (i.e., contacting) a collagen tube with cells and culturing the seeded tube under conditions suitable for growth of the cells. Examples of growth conditions include use of particular growth media and application of mechanical/electrical/chemical stimuli. The cells can be derived from an animal or a cell line of the same species as the intended recipient so that the resulting tube contains proteins that are minimally antigenic and maximally compatible in the recipient. For example, if the collagen tube is to be implanted into a human, the cells are preferably human cells.

The collagen tube of this invention can be implanted into a patient to replace or bypass abnormal arteries and veins in conditions such as aneurysm and peripheral artery disease, or atherosclerosis or arteries and veins that are or blocked or damaged by injury or vascular disease, e.g., thrombosis or stroke.

The collagen tube can also be used in nerve repairing. The linear nature of the collagen fibers provides an ideal structure to guide the growth of the neural cells.

Further, the collagen tube can be used in urethral replacement or used as bioartificial renal tubular device in treating renal failure.

Without further elaboration, it is believed that one skilled in the art can, based on the disclosure herein, utilize the present invention to its fullest extent. The following specific examples are, therefore, to be construed as merely descriptive, and not limitative of the remainder of the disclosure in any way whatsoever. All publications and patent documents cited herein are incorporated by reference. Preparing Multi-layered Collagen Tube of This Invention

Tendon was obtained from cow tendon or rat tail and cut into 10 x 10 x 3 mm blocks. The blocks were then placed into a surfactant solution (Tris buffer, 5 mM EDTA and 1% SDS) and placed on a shaker at 4°C for 48 h to remove cells. The tendon blocks were placed in deionized H₂0 for 24 h at 4°C, and then removed from H₂0, embedded in OCT media, and frozen at -20°C. The frozen blocks were sectioned with a cryostat microtome into slices having a thickness of 20-100 μιη depending on the desired properties. These square sections were stacked with alternating fiber orientations in groups of 2-10. The stacks were wrapped around a glass capillary tube and allowed to dry overnight.

Once dried, the tubular sections were removed. They were optionally immersed into a prepolymer solution of poly(dimethylsiloxane) and the resulting tubes were removed and dried. Figures 3 and 4 respectively show the SEM and optical images of the wall of a thus-obtained collagen tube.

Assessing Biomechanical Properties and Cell Viability of the Collagen Tube

Collagen fiber tubes were cut into pieces of approximately 15 mm in length and 8 mm in width. Each piece was placed onto an Instron 3366 uniaxial material testing device equipped with a 100 N load cell. It was then subjected to a tensile stress at a strain rate of 0.5 mm/min until failure. The load-displacement data was collected and converted to stress-strain data based on initial dimensional measurements to ensure normalized comparison between samples of different sizes. Various sample configurations and orientations were tested to determine the structure having the optimal mechanical properties. The samples that displayed the optimal mechanical properties were then replicated and subjected to a hydrated testing. The samples were hydrated in phosphate buffered saline (PBS) for one hour prior to testing. An Instron testing apparatus was modified with a water bath filled with PBS. The testing was repeated and the data collected and analyzed in the same way as described above. The optimal biomechanical properties were then compared with cell viability data to determine the best scaffold construction technique.

Three types of collagen fiber tubes were tested: (i) collagen fiber tubes having collagen fibers oriented in alternating directions as described above (crossed sections), (ii) collagen fiber tubes having all of the fibers aligned in the same direction and having the tensile force applied parallel to that direction (linear sections), and (iii) collagen fiber tubes having all of the fibers aligned in the same direction and having the tensile force applied perpendicular to that direction (antilinear sections). As shown in Figure 5, the collagen fiber tubes of this invention had preferred stress-strain properties. More specifically, crossed sections (i) had mechanical strength than linear sections (ii) and antilinear sections (iii).

Collagen fiber tubes of this invention were also used to grow PC-2 neuron cells. Beef tendon was obtained and decellularized by extraction with Tris-HCl buffer, 5 mM EDTA, and 1% (w/v) SDS. The decellularized tendon was fixed in 4%

formaldehyde and immersed in ethanol solutions with the gradually increasing ethanol concentrations for dehydration. The dehydrated tendon was either embedded into paraffin for traditional microtome sectioning, or sectioned directly without cryomicrotome. The tendon was cut along the longitudinal alignment of the collagen fibers, which were then transferred to a glass slide for further processing. The tendon slices were immersed in xylene to remove paraffin (when needed). All of the tendon slices were rehydrated by immersion in ethanol solutions with the gradually decreasing ethanol concentrations.

The rehydrated slices were used as scaffolds for aligned and directionally guided nerve growth as follows. Cells of the PC 12 cell line, obtained from ATCC, were seeded onto the tendon slices with a density of 5 x 10⁴ cells per well, or 1.3 x 10⁴ cells/cm² in custom culture medium (89% v/v Dulbecco's Modified Eagle Medium, 5% v/v horse serum, 5% v/v fetal bovine serum, and 1% v/v Penicillin: Streptomycin solution). The cells were incubated at 37°C for ~24 hours not in any differentiation medium. The growth medium was then replaced with custom differentiation medium (97% v/v DMEM, 1% v/v Nerve Growth Factor, 1% v/v Horse Serum, 1% v/v Penicillin: Streptomycin solution). The cells were allowed to grow for 1 week, during which the medium was replaced every two days.

The tendon slices were then removed from the medium, and fluorescence stained with 25 μϊ_^ of Texas Red-x phalloidin in methanol and one drop of 4',6-diamidino-2- phenylindole solution per tendon. They were analyzed using light fluorescence microscopy. Scanning electron microscopy was used to image the tendon slices in greater detail. As shown in Figure 6, PC-2 neuron cells grew linearly along the collagen fibers in the tube wall.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, a person skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the present invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

Claims

WHAT IS CLAIMED IS:

1. A collagen scaffold comprising 2-10 stacked collagen fiber layers, wherein at least 90% of the collagen fibers in each layer have a length of at least 2 mm and are so oriented that they extend along the layer and are parallel to each other, each collagen fiber layer has a thickness of 10-100 μιη and features a direction identical to the orientation of the at least 90% of the collagen fibers, and the angle between the directions of every collage fiber layers next to each other is 30°-90°.

2. The collagen scaffold of claim 1, further comprising a polymer coating on the surface of the collagen scaffold.

3. The collagen scaffold of claim 1, wherein the angle between the directions of every collage fiber layers next to each other is 45°-90°.

4. The collagen scaffold of claim 1, wherein the angle between the directions of every collage fiber layers next to each other is 60°-90°.

5. The collagen scaffold of claim 1, wherein the angle between the directions of every collage fiber layers next to each other is 80°-90°.

6. The collagen scaffold of claim 1, further comprising 1-9 non-collagen elastic layers, wherein each of the elastic layers is deposited between two collagen fiber layers next to each other.

7. The collagen scaffold of claim 6, wherein the elastic layers are aorta vessel wall slices.

8. The collagen scaffold of claim 6, wherein the elastic layers are synthetic polymer slices.

9. The collagen scaffold of claim 6, wherein the angle between the directions of every collage fiber layers next to each other is 45°-90°.

10. The collagen scaffold of claim 9, wherein the angle between the directions of every collage fiber layers next to each other is 80°-90°.

11. A collagen tube comprising a tubular wall including 2-10 stacked collagen fiber layers, wherein at least 90% of the collagen fibers in each layer have a length of at least 2 mm and are so oriented that they extend along the layer and are parallel to each other, each layer has a thickness of 10-100 μιη and features a direction identical to the orientation of the at least 90% of the collagen fibers, and the angle between the directions of every collage fiber layers next to each other is 30°-90°.

12. The collagen tube of claim 1 1, further comprising a polymer coating on the external surface of the tubular wall.

13. The collagen tube of claim 11, wherein the angle between the directions of every collage fiber layers next to each other is 45°-90°.

14. The collagen tube of claim 11, wherein the angle between the directions of every collage fiber layers next to each other is 60°-90°.

15. The collagen tube of claim 11, wherein the angle between the directions of every collage fiber layers next to each other is 80°-90°.

16. The collagen tube of claim 11, further comprising 1-9 non-collagen elastic layers, wherein each of the elastic layers is deposited between two adjacent collagen fiber layers next to each other.

17. The collagen tube of claim 16, wherein the elastic layers are aorta vessel wall slices.

18. The collagen tube of claim 16, wherein the elastic layers are synthetic polymer slices.

19. The collagen tube of claim 16, wherein the angle between the directions of every collage fiber layers next to each other is 45°-90°.

20. The collagen tube of claim 9, wherein the angle between the directions of every collage fiber layers next to each other is 80°-90°.

21. A method of producing a collagen tubular structure comprising:

providing layers of collagen fibers, wherein at least 90% of the collagen fibers in each layer are so oriented that they extend along the layer and are parallel to each other, and each layer has a thickness of 10-100 μιη and features a direction defined by the orientation of the at least 90% of the collagen fibers;

stacking the layers of collagen fibers to form a multilayer sheet in such a manner that the angle between directions of any two layers next to each other in the multilayer sheet is 30°-90°; and

wrapping the multilayer sheet around a cylindrical object having a diameter of

0.1-10 mm and removing the cylindrical object to provide a collagen tube.

22. The method of claim 21, wherein the layers of collagen fibers are obtained from tendon, bone, skin, or ligament by cryostat microtome.

23. The method of claim 21, further comprising immersing the collagen tube in a polymer solution so as to form a polymer coating on the collagen tube.

24. The method of claim 23, wherein the polymer solution further comprising a biologically active agent.

25. The method of claim 21, wherein, in the stacking step, the angle between directions of every two adjacent in the multilayer sheet is 60°-90°.

26. The method of claim 21, wherein, in the stacking step, the angle between directions of every two adjacent in the multilayer sheet is 80°-90°.