Multilayer collagen structure

Abstract

It is biocompatible and capable of augmenting both soft and hard tissue, providing a collagen structure that retains each type of tissue in the desired area. A layered structure is provided that includes at least two collagen layers having different densities.

Description

[Technical Field]

[0001] The present invention relates to a multi-layer collagen structure comprising at least two layers with different densities. The present invention further relates to a method for preparing such a multi-layer structure, comprising compressing fibrillated collagen in the presence of at least one separating element. [Background technology]

[0002] Guided bone regeneration (GBR) and guided tissue regeneration (GTR) are known procedures for bone and tissue augmentation. Both GBR and GTR require the exclusion of unwanted tissues from occupying the space into which the regenerated bone or tissue will grow. This isolated space allows for the proliferation, migration, and differentiation of bone-forming cells, such as bone-derived cells, mesenchymal stem cells, gingival cells, and fibroblasts. This isolated space must be maintained to allow the new bone to mature and stabilize with minimal resorption. It has been shown that the highest success rate of GBR procedures is achieved by combining bone graft tissue with a barrier membrane that prevents unwanted tissues from occupying the space into which the bone will regenerate.

[0003] In many cases, the bone covering the dental implant is not ideal and does not provide long-term protection. Peri-implantitis, which resembles periodontal disease around natural teeth, often develops. In other instances, the esthetics of the implant procedure are compromised due to reflection of the implant through the bone and / or soft tissue, or due to soft tissue recession exposing the metallic components of the implant.

[0004] Harvesting autologous dense connective tissue, for example from the palate or tuberosity, allows for oral soft tissue augmentation as needed, for example for aesthetic reasons. Allografts have also been widely used with limited results. However, such procedures require additional surgical procedures, can cause pain and infection, and can be rejected by the body. Therefore, there is a need in the art for biocompatible and, whenever possible, at least biodegradable devices for augmenting the soft and hard tissues around natural teeth and dental implants. Such devices should be made from biocompatible and, whenever possible, biodegradable materials.

[0005] One type of known biocompatible and biodegradable material is collagen. Collagen protein accounts for approximately 30% of the proteins in the body and plays a role in supporting the adhesion between bone and cells. Therefore, collagen is known to be a useful biomaterial, used, for example, in substrates for cultured cells and as a scaffolding material for regenerative medicine, including tissue engineering of cartilage, bone, ligament, corneal stroma, and skin. Collagen is also used as a transplant material, for example, as a wound dressing material, bone graft material, hemostatic material, or anti-adhesion material.

[0006] Therefore, it is desirable to prepare a collagen structure that is biocompatible and yet capable of augmenting both soft and hard tissue, with each type of tissue remaining in the desired area. Summary of the Invention [Means for solving the problem]

[0007] The present invention provides a layered structure comprising at least two collagen layers, each having a different density.

[0008] In some embodiments, the present invention provides a layered structure comprising at least two collagen layers, each having a different density, the at least two collagen layers comprising at least one first collagen layer and at least one second collagen layer, wherein the at least one second collagen layer (low density layer) has a density up to about 90% of the density of the first collagen layer (high density layer).

[0009] In some embodiments, the present invention provides a layered structure comprising at least two collagen layers, each having a different density, the at least two collagen layers comprising at least two first collagen layers and at least one second collagen layer, the at least one second collagen layer (inner layer) being disposed between each of the at least two first collagen layers (outer layers).

[0010] In some embodiments, the present invention provides a layered structure comprising at least two collagen layers, each having a different density, the at least two collagen layers comprising at least two first collagen layers and at least one second collagen layer, the at least one second collagen layer (inner layer) being disposed between each of the at least two first collagen layers (outer layers), and the at least one second collagen layer (low density layer) each independently having a density of up to about 90% of the density of the at least two first collagen layers.

[0011] In some embodiments, the present invention provides a layered structure comprising at least two collagen layers, each having a different density, the at least two collagen layers comprising at least two first collagen layers and at least one second collagen layer, the at least one first collagen layer (inner layer) being disposed between each of the at least two first collagen layers (outer layers), and the at least two first collagen layers (low density layers) each independently having a density of up to about 90% of the density of the at least one second collagen layer (high density layer). [Brief explanation of the drawings]

[0012] The subject matter which is regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of this specification. The invention, however, together with its objects, features, and advantages, both as to organization and method of operation, may best be understood by reference to the following detailed description when read in connection with the accompanying drawings, in which: Embodiments of the invention are illustrated by way of example, and not by way of limitation, in the accompanying drawings in which like reference numerals indicate corresponding, similar, or similar elements.

[0013] [Figure 1] FIG. 1 is a photographic image of a three-layer collagen structure according to an embodiment of the present invention. [Figure 2] FIG. 2 is a 2x magnification view of a three-layer collagen structure according to an embodiment of the present invention. [Figure 3] FIG. 1 is a photomicrograph of a layered structure implant of the present invention in a dog four weeks after implantation, implanted to repair an L-shaped body defect, the collagen structure is marked with an "M," showing the beginning of ossification of the multilayered collagen structure. [Figure 4] FIG. 1 is a photomicrograph of a graft in a dog 24 weeks after implantation, which was transplanted to repair an L-shaped body defect. [Figure 5] Photomicrograph of a graft in a dog 4 weeks after implantation, implanted to repair an L-shaped corporal defect, showing two outer layers and one inner layer of the multilayered collagen graft structure. [Figure 6A] FIG. 1 illustrates the preparation of a structure of the present invention without the use of a spacer. [Figure 6B] 10A-10C illustrate the preparation of the structure of the present invention when using spacers. [Figure 7A] 1 shows a layered structure of the present invention with five layers of different density: Electron microscope images (low resolution): cross section of the multilayer film, #1, #5: outer layers which are dense layers of the multilayer film, #2, #3, #4: inner layers which are less dense layers of the multilayer film. [Figure 7B] Electron microscope images (high resolution): Comparison of low-density and high-density layers of the multilayer membrane (high-density and low-density aspects of collagen fibers). DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention provides a layered structure comprising at least two collagen layers, each having a different density.

[0015] The term "layered structure" should be understood to include a collection of at least two collagen layers arranged on top of each other. The layered structure of the present invention comprises at least two collagen layers, each of which has a density different from the other layer. This term is used interchangeably with the terms "collagen layered structure" and "collagen layered membrane." The layered structure can be part of a device, matrix, or scaffold.

[0016] Collagen is the major structural protein in the extracellular matrix of various connective tissues in the body and consists of amino acids linked together to form triple helices of elongated fibers known as collagen helices, which are found primarily in connective tissues and membranes such as cartilage, bone, tendons, ligaments, and skin.

[0017] The collagen layer may have different densities depending on the degree of mineralization. In some embodiments, the collagen layer may be stiff (e.g., in bone), compliant (e.g., in tendon), or have a gradient from stiff to compliant (e.g., in cartilage).

[0018] In some embodiments, the layered structure is a membrane, hi other embodiments, the layered structure is an implantable membrane.

[0019] Soft and / or hard tissues may be enhanced differently with each collagen layer of the layered structure of the present invention due to the difference in density of each collagen layer. According to some embodiments, the regenerated tissue is maintained for more than six months.

[0020] According to some embodiments, the layered structure of the present invention is used as a surgical scaffold for soft tissue growth / augmentation (e.g., dorsal augmentation). According to further embodiments, the layered structure of the present invention is used to reinforce soft tissue as needed, for example, during hernia repair. According to further embodiments, the layered structure of the present invention is used in treatments involving bone growth / augmentation (e.g., guided bone regeneration (GBR) and guided tissue regeneration (GTR) in the treatment of periodontal defects). The layered structure of the present invention may also be used for soft tissue growth / augmentation, such as gum augmentation, face lift / repair, and soft tissue and cartilage lift / augmentation, for example, of the nose or ear. According to some embodiments, the layered structure of the present invention is used in any combination of soft and hard tissue growth / augmentation, depending on the area in which the membrane is placed.

[0021] According to some embodiments, the layered structures of the present invention are used to prevent adhesions in tissues and organs. Prevention of adhesions occurs by using layered structures of the present invention that include a relatively low-density inner collagen layer that can degrade at a faster rate than the denser outer collagen layer, such that upon degradation the layered structures of the present invention essentially separate into two separate membranes, thereby preventing adhesions between tissue above and below the layered structures of the present invention.

[0022] According to some embodiments, the layered structures of the present invention provide long-term protection and aesthetics to teeth and dental implants and other augmentation sites. As detailed herein, the implanted layered structures of the present invention can partially or completely densify, degrade, and be replaced by surrounding tissue.

[0023] According to some embodiments, one or more layers of the layered structure of the present invention promote the proliferation of various cell types. According to some embodiments, the promotion of various cell types depends on the density of the layer. Thus, specific regions of the layered structure of the present invention promote the growth of specific types of tissue, including gingival tissue and bone tissue.

[0024] According to some embodiments, bone tissue growth is promoted with a relatively dense collagen layer, while soft tissue growth is promoted with a relatively less dense collagen layer.

[0025] According to some embodiments, the layered structure of the present invention comprises a relatively low-density collagen layer sandwiched between relatively high-density collagen layers such that the high-density collagen layer is exposed to surrounding tissue. According to some embodiments, the high-density collagen layer promotes bone tissue growth, while the low-density collagen layer promotes soft tissue growth. According to some embodiments, fibroblasts can penetrate the inner collagen layer, for example, through at least one high-density collagen layer of the layered structure of the present invention and / or under / over / around at least one high-density collagen layer of the layered structure of the present invention, and grow soft tissue into the inner low-density collagen layer.

[0026] In some embodiments, the terms "high density collagen layer," "relatively high density collagen layer," and the like refer to a collagen layer having a density of about 1.41 g / cm 3 Furthermore, terms such as "low density collagen layer," "low density collagen layer," and "relatively low density collagen layer" refer to a layer having an average density of about 1.17 g / cm. 3 The term "about" defines a layer having an average density of about 1.0-1.7 g / cm. Additionally, the term "about" includes a range of ±10% of the disclosed value. According to some embodiments, the density of the relatively dense collagen layer is about 1.0-1.7 g / cm. 3 According to some embodiments, the density of the relatively dense collagen layer is in the range of about 1.0-2 g / cm 3 According to some embodiments, the density of the relatively low density collagen layer is in the range of about 0.4 to 1.0 g / cm 3 The range is.

[0027] In some embodiments, the density is determined by Hg porosimetry.

[0028] According to some embodiments, the layered structure of the present invention comprises at least two collagen layers, at least one collagen layer having a relatively low density and at least one collagen layer having a relatively high density, the relatively low density collagen layer having a density that is up to about 90% of the density of the high density collagen layer. According to some embodiments, the relatively low density collagen layer has a density that is up to about 85% of the density of the high density collagen layer. According to some embodiments, the relatively low density collagen layer has a density that is up to about 80% of the density of the high density collagen layer. According to some embodiments, the relatively low density collagen layer has a density that is up to about 75% of the density of the high density collagen layer. According to some embodiments, the relatively low density collagen layer has a density that is up to about 70% of the density of the high density collagen layer.

[0029] According to some embodiments, the relatively low-density collagen layer has a density between 10% and 90% of the high-density collagen layer. According to some embodiments, the relatively low-density collagen layer has a density between 20% and 90% of the high-density collagen layer. According to some embodiments, the relatively low-density collagen layer has a density between 30% and 90% of the high-density collagen layer. According to some embodiments, the relatively low-density collagen layer has a density between 40% and 90% of the high-density collagen layer. According to some embodiments, the relatively low-density collagen layer has a density between 50% and 90% of the high-density collagen layer. According to some embodiments, the relatively low-density collagen layer has a density between 60% and 90% of the high-density collagen layer. According to some embodiments, the relatively low-density collagen layer has a density between 70% and 90% of the high-density collagen layer. According to some embodiments, the relatively low-density collagen layer has a density between 80% and 90% of the high-density collagen layer.

[0030] In some embodiments, the present invention provides a layered structure comprising at least two collagen layers, each having a different density, the at least two collagen layers comprising at least one first collagen layer and at least one second collagen layer, wherein the at least one second collagen layer (low density layer) has a density up to about 90% of the density of the first collagen layer (high density layer).

[0031] In some embodiments, the present invention provides a layered structure comprising at least two collagen layers, each having a different density, the at least two collagen layers comprising at least two first collagen layers and at least one second collagen layer, the at least one second collagen layer (inner layer) being disposed between each of the at least two first collagen layers (outer layers).

[0032] In some embodiments, the present invention provides a layered structure comprising at least two collagen layers, each having a different density, the at least two collagen layers comprising at least two first collagen layers and at least one second collagen layer, the at least one second collagen layer (inner layer) being disposed between each of the at least two first collagen layers (outer layers), and the at least one second collagen layer (low density layer) each independently having a density of up to about 90% of the density of the at least two first collagen layers (high density layers).

[0033] In some embodiments, the present invention provides a layered structure comprising at least two collagen layers, each having a different density, the at least two collagen layers comprising at least two first collagen layers and at least one second collagen layer, the at least one second collagen layer (inner layer) being disposed between each of the at least two first collagen layers (outer layers), and the at least two first collagen layers (low density layers) each independently having a density of up to about 90% of the density of the at least one second collagen layer (high density layer).

[0034] In some embodiments, the at least two collagen layers include at least one first collagen layer and at least one second collagen layer, and the at least one first collagen layer has a density that is at least 90% greater than the density of the at least one second collagen layer.

[0035] In some further embodiments, the at least two first collagen layers independently have a density that is at least 90% greater than the density of the at least one second collagen layer.

[0036] In some further embodiments, the at least one second collagen layer has a density that is at least 90% greater than the density of each of the at least two first collagen layers.

[0037] According to some embodiments, the layered structures of the present invention comprise two, three, four, or five collagen layers. According to some embodiments, the layered structures of the present invention comprise multiple layers, each having a different density. According to some embodiments, the layered structures of the present invention do not comprise distinct, separate layers, but rather comprise a density gradient, such that at least one section of the device has a different, and possibly varying, density than another section of the device. According to some embodiments, the densities of the various sections are between 0.4 and 2 g / cm. 3 According to some embodiments, the density of the relatively dense collagen layer is in the range of about 1.0 to 1.7 g / cm 3 The range is.

[0038] According to some embodiments, the layered structure of the present invention comprises three collagen layers, the outer layer being dense and the inner layer being relatively low density. In some embodiments, the inner layer may be a gel-like layer. According to some embodiments, the denser outer layer has a different thickness than the inner layer. In some embodiments, the thickness of the inner layer is greater than the thickness of the outer layer.

[0039] According to some embodiments, the inner layer has a thickness of about 0.5 to 5.0 mm. According to some embodiments, the inner layer has a thickness of about 0.5 to 1.0 mm. According to some embodiments, the inner layer has a thickness of about 1.0 to 2.0 mm. According to some embodiments, the inner layer has a thickness of about 2.0 to 3.0 mm. According to some embodiments, the inner layer has a thickness of about 3.0 to 4.0 mm. According to some embodiments, the inner layer has a thickness of about 4.0 to 5.0 mm.

[0040] According to some embodiments, the outer layer has a thickness of about 0.05 to 0.4 mm. According to some embodiments, the outer layer has a thickness of about 0.05 to 0.1 mm. According to some embodiments, the outer layer has a thickness of about 0.1 to 0.2 mm. According to some embodiments, the outer layer has a thickness of about 0.2 to 0.3 mm. According to some embodiments, the outer layer has a thickness of about 0.3 to 0.4 mm.

[0041] Embodiments of the layered structure of the present invention are shown, for example, in Figure 1 (a photographic image at 1x magnification) and Figure 2 (a cross-section of the device at 2x magnification). According to some embodiments, the average density of the outer layer is about 1.41 g / cm 3 According to some embodiments, the average density of the inner layer is about 1.17 g / cm 3 According to some embodiments, the density of the outer layer is about 1.0 to 1.7 g / cm 3 According to some embodiments, the density of the inner layer is in the range of about 0.4 to 1.0 g / cm 3 The range is.

[0042] An embodiment of the present invention relates to a method for preparing a layered structure of the present invention, the method comprising the steps of introducing collagen into a container, fibrillating the collagen to provide a collagen gel comprising collagen fibrils (collagen fibrils), compressing the collagen gel in the presence of at least one separating element to provide at least two layers with different densities, cross-linking the collagen gel, and drying the cross-linked collagen.

[0043] According to some embodiments, collagen is fibrillated using a fibrillation agent. Collagen may be fibrillated by any method known in the art. According to some embodiments, collagen is fibrillated by neutralizing its pH, for example, by mixing it with a buffer solution having a neutral or basic pH. According to some embodiments, the fibrillation agent includes one or more bases and / or salts, such as sodium phosphate, Tris-HCl, potassium hydroxide, or sodium hydroxide.

[0044] Once the collagen is fibrillated, it can be at least partially compressed in the presence of a separating element to provide at least two layers with different densities. According to some embodiments, the collagen is compressed so that layers are formed. For example, when the collagen is compressed, its outer layer is compressed more than the inner layer, and the collagen, particularly the inner layer, is not fully compressed due to the presence of the separating element. Thus, compression can be stopped at any suitable point, for example, to form a relatively dense layer on the outside of the device and a relatively less dense layer on the inside of the device.

[0045] According to some embodiments, at least one of the outer layers is more compressed than the inner layer. According to some embodiments, the two outer layers are more compressed than the one inner layer, thereby providing a three-layer collagen component having two relatively high-density outer layers and one relatively low-density inner layer. According to some embodiments, the two outer layers are more compressed than the one inner layer, thereby providing a three-layer collagen component with a first relatively high-density outer layer, a second outer layer having a lower density than the first outer layer, and one relatively low-density inner layer. According to some embodiments, the compression provides a density gradient ranging from a high density in the outer layers of the layered structure of the present invention to a lower density in the inner layers. The two outer layers may have similar densities (about a 10% difference) or different densities, but each has a higher density than the inner layer.

[0046] As described above, collagen is compressed in the presence of at least one separating element, e.g., a weight, that prevents the compressing element from fully compressing the fibrillated collagen. Referring to Figures 6A and 6B, compression of fibrillated collagen is shown in the absence (Figure 6A) and presence (Figure 6B) of a separating element. As shown, the separating element creates a distance between the compressing element and the bottom of the reaction vessel, thereby preventing the compressing element from fully compressing the fibrillated collagen. Thus, as shown, when compressed in the presence of a separating element, the multilayer structure of the present invention prepared is thicker than the layer structure of the present invention prepared without the use of a separating element. Note that the final thickness of the multilayer structure of the present invention depends at least in part on the height of the separating element.

[0047] According to some embodiments, the height of the separation element is about 0.1 to 10 mm. According to some embodiments, the height of the separation element is about 0.1 to 1.0 mm. According to some embodiments, the height of the separation element is about 1.0 to 2.0 mm. According to some embodiments, the height of the separation element is about 2.0 to 3.0 mm. According to some embodiments, the height of the separation element is about 3.0 to 4.0 mm. According to some embodiments, the height of the separation element is about 4.0 to 5.0 mm. According to some embodiments, the height of the separation element is about 5.0 to 6.0 mm. According to some embodiments, the height of the separation element is about 6.0 to 7.0 mm. According to some embodiments, the height of the separation element is about 7.0 to 8.0 mm. According to some embodiments, the height of the separation element is about 8.0 to 9.0 mm. According to some embodiments, the height of the separation element is about 9.0 to 10 mm.

[0048] According to some embodiments, the separation element is prepared from any suitable material that can provide the desired distance without interfering with the collagen preparation process, such as Teflon, stainless steel, silicone, polypropylene, polyethylene, or any combination thereof. According to some embodiments, the separation element is fixed within the reaction vessel. According to some embodiments, the separation element is positioned vertically within the reaction vessel. According to some embodiments, the separation element is provided in the reaction vessel at any stage before compression. According to some embodiments, the separation element may be provided in the reaction vessel at any stage during compression, as long as the fibrillated collagen is not over-compressed before the separation element is provided. According to other embodiments, the fibrillated collagen may be transferred to the reaction vessel with the separation element before compression. Once compression is complete, the separation element is separated from the prepared multilayer structure of the present invention by removing the separation element from the reaction vessel or by removing the multilayer structure of the present invention from the reaction vessel.

[0049] Once at least partially compressed, the collagen is cross-linked by any method known in the art. According to some embodiments, the compressed collagen gel is contacted with a cross-linking agent.

[0050] According to some embodiments, the collagen is crosslinked by an enzyme-mediated process, by physical treatment (e.g., heat, UV radiation), or by a chemical crosslinker, wherein the crosslinker comprises a reducing agent such as a reducing sugar or a reducing sugar derivative, or any combination thereof.

[0051] According to some embodiments, the crosslinker comprises an aldehyde or ketone monosugar or monosugar derivative, wherein the α-carbon is in the aldehyde or ketone state in aqueous solution. According to some embodiments, the crosslinker comprises the compounds and reagents detailed in U.S. Pat. No. 6,346,515, which is incorporated herein by reference. As detailed in U.S. Pat. No. 6,346,515, a reducing sugar can form a Schiff base with the α- or ε-amino group of an amino acid in a collagen molecule. The Schiff base then undergoes an Amadori rearrangement to form a ketoamine product. Two adjacent ketoamine groups then condense to form stable inter- or intramolecular crosslinks.

[0052] Reducing agents include, for example, glycerose, threose, erythrose, lyxose, xylose, arabinose, ribose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose or any other diose, triose, tetrose, pentose, hexose, heptose, octose, nonose, decose, or any combination thereof. For example, when the cross-linking agent contains ribose, stable cross-links can be formed via pentosidine groups.

[0053] According to some embodiments, the degradation rate of the layered structure of the present invention is controlled by the degree of crosslinking between collagen fibrils. The degree of crosslinking is controlled, for example, by the concentration of the crosslinking agent, the temperature, and the time the collagen fibrils are exposed to the crosslinking agent. According to some embodiments, crosslinking is carried out at a crosslinking agent concentration ranging from about 0.01% to 5%. According to some embodiments, crosslinking is carried out at a temperature ranging from about 20 to 40°C. According to some embodiments, crosslinking is carried out for a duration ranging from about 6 to 360 hours.

[0054] According to some embodiments, the tissue surrounding the layered structure of the present invention regenerates, at least partially, over time to replace the layered structure of the present invention. Thus, the layered structure of the present invention provides a space for the growth of surrounding tissue. According to some embodiments, the separation elements used in the process for preparing the layered structure of the present invention provide the layered structure of the present invention with voids between collagen molecules because the collagen was not fully compressed during device preparation. The voids formed allow the surrounding tissue to infiltrate the formed spaces and grow into them over time, replacing the degrading collagen. According to some embodiments, the layered structure of the present invention mineralizes and subsequently ossifies, providing a foundation for a bone cell population.

[0055] According to some embodiments, the layered structures of the present invention are biodegradable. According to some embodiments, the layered structures of the present invention are biodegraded within about 3-24 months. According to some embodiments, the layered structures of the present invention are biodegraded within about 4-8 months. According to some embodiments, the layered structures of the present invention are biodegraded within about 5-7 months. According to some embodiments, the layered structures of the present invention are biodegraded within about 3-10 months. According to some embodiments, the layered structures of the present invention are biodegraded within about 10-17 months. According to some embodiments, the layered structures of the present invention are biodegraded within about 17-24 months. According to some embodiments, the layered structures of the present invention are biodegraded within about 6 months. It should be noted that the layered structures of the present invention are considered biodegraded when they are greater than about 70, 80, 90, or 95% degraded.

[0056] According to some embodiments, different layers of the layered structure of the present invention degrade at different rates depending on their density, with denser layers degrading at slower rates than less dense layers, according to some embodiments.

[0057] According to some embodiments, once the collagen is crosslinked, the prepared layered structure of the present invention is washed to remove residual reactants, such as fibrillating agents, crosslinking agents, and uncrosslinked collagen. According to further embodiments, the collagen film is then at least partially dehydrated by any suitable means, such as air drying, freeze-drying, critical point drying, or any combination thereof. The drying process further sterilizes and dries the multilayered structures of the present invention, effectively extending their shelf life. According to some embodiments, the layers of the dehydrated multilayered structures of the present invention are less visible, but according to some embodiments, rehydration with any suitable liquid, such as water, saline, blood, or phosphate-buffered saline (PBS) solution, restores the three-dimensional structure of the multilayered structures of the present invention. According to some embodiments, the multilayered structures of the present invention are rehydrated in vitro. According to other embodiments, the multilayered structures of the present invention are rehydrated in vivo.

[0058] According to some embodiments, the layered structures of the present invention contain active agents with various therapeutic effects. In some embodiments, the active agents are immobilized within the layered structures of the present invention by crosslinking agents, such as reducing sugars, or by their inherent tendency to bind to collagen. During the gradual biodegradation of the layered structures of the present invention, such active agents are gradually released into the body, thereby providing a controlled release system. Such active agents include antimicrobial agents, anti-inflammatory agents, growth factors with tissue regeneration-inducing properties, and any combination thereof.

[0059] Antibacterial agents may include penicillin, cephalosporins, tetracyclines, streptomycin, gentamicin, sulfonamides, and miconazole. Anti-inflammatory agents may include cortisone, its synthetic derivatives, etc. Tissue regeneration inducers may include differentiation factors, bone morphogenetic proteins, attachment factors, and growth factors, such as fibroblast growth factors, platelet-derived growth factors, transforming growth factors, cementum growth factors, insulin-like growth factors, etc.

[0060] According to some embodiments, additional biodegradable materials may be added, such as polylactic acid (PLA), polyglycolide (PGA), chitosan, hyaluronic acid, and mixtures thereof. According to further embodiments, any type of mineral may be added, such as tricalcium phosphate, silicon oxide, hydroxyapatite, and the like.

[0061] According to some embodiments, cells such as human cells, bone cells, cultured cells, fibroblasts, chondrocytes, (mesenchymal) stem cells, osteocytes, adipocytes, endothelial cells, etc., are seeded on or in at least one of the layers, such as the outer dense layer, which is to be arranged in the direction of the bone. Such a layer functions as a protective semi-permeable layer to provide the possibility of supplying fluids containing nutrients, e.g., minerals, to the inner layers of the device and / or tissues found below the device, protecting the inner layers of the device and / or tissues below the device from direct interaction with other cell types. According to some embodiments, interaction of the inner layers and / or tissues below the device is prevented due to the pore size of at least one layer in the multilayer structure of the present invention, which is smaller than the pore size required for the passage of certain cell types.

[0062] According to some embodiments, the multi-layer structures of the present invention can be used in combination with space-maintaining materials ("space maintainers" or "spacers"). The term "in combination" is intended to encompass uses in which the space maintainer is adjacent to the multi-layer structure of the present invention, attached to the multi-layer structure of the present invention by any suitable means, or incorporated into the multi-layer structure of the present invention.

[0063] Space maintainers are used in some procedures to maintain space into which regenerative cells can migrate and regrow. In some instances, such spaces occur naturally, for example, when a tumor is excised from bone. In other instances, such as various types of periodontal or bone injuries, such spaces do not occur. In such cases, a filler must be inserted between the collagen membrane and the regenerated tissue. Examples of space maintainers include: (i) hyaluronan (hyaluronic acid), (ii) mineralized freeze-dried bone, (iii) deproteinized bone, (iv) synthetic hydroxyapatite, (v) crystalline materials rich in osteocalcin or vitronectin other than those mentioned in (ii)-(iv), and (vi) heat-treated demineralized bone, in which the bone-derived material is of human origin. Combinations of any of the above space maintainers may also be used, such as a combination of hyaluronan with one or more other space maintainers.

[0064] For various applications depending on the size, shape, and location of the regeneration site, the space maintainer may be enriched with one or more of the above-mentioned antimicrobial, anti-inflammatory, and tissue induction factors and / or enriched with substances intended to help maintain the shape of the space maintainer matrix, such as one or more matrix proteins selected from the group including collagen, fibrin, fibronectin, osteonectin, osteopontin, tenascin, thrombospondin, and / or glycosaminoglycans, including heparin sulfate, dermatan sulfate, chondroitin sulfate, keratan sulfate, and the like.

[0065] According to some embodiments, the layered structures of the present invention are designed to fill the space where tissue is to be regenerated. Thus, the use of a space maintainer is not necessary as the tissue regenerates and is replaced by the biodegradable layered structures of the present invention. According to some embodiments, the layered structures of the present invention are prepared to have any size or shape, as detailed herein.

[0066] Referring to Figures 3-5, photomicrographs are shown 4 weeks after implantation in an L-shaped bone defect in a dog. Note the early ossification progressing in the middle layer of the device (arrows). The initial collagen matrix of the device is marked (M) (Figure 3).

[0067] Figure 4 shows a photomicrograph of a canine L-shaped bone defect 24 weeks after implantation. Note the progression of ossification in the middle layer of the device (arrow).

[0068] Figure 5 shows a micrograph of a canine L-shaped bone defect four weeks after implantation. The membrane has a characteristic three-layer structure surrounded by soft tissue (*: low density, arrow: high density).

[0069] According to some embodiments, the multi-layer structure of the present invention comprises a scaffold, as detailed in US Pat. No. 62 / 317,569, which is incorporated herein by reference.

[0070] To better understand how the present invention may be practiced, the following examples are provided to demonstrate processes according to the present disclosure.

[0071] Example

[0072] Example 1

[0073] A 320 ml aliquot of collagen was mixed with 30 ml of fibrillation buffer consisting of 200 mM sodium phosphate at pH 11.2. After mixing with the fibrillation buffer, the collagen was poured into an 11 x 16 cm forming plate containing a 3 mm high Teflon-based separating element to maintain the desired final membrane thickness. Fibrillation was allowed to proceed for 18 hours at 37°C. The resulting gel was compressed for 20 hours at 37°C using a 1 kg weight, with the separating element ensuring a distance of at least 3 mm between the weight and the bottom of the forming plate during compression. The collagen sheet was crosslinked for 11 days at 37°C in a 0.2 micron filtration medium of 10 g ribose, 158 g ethanol, and 790 g PBS. The resulting membrane was then washed with water, dehydrated with ethanol, and dried by lyophilization. FIG. 1 shows the prepared wet collagen membrane at low magnification, and FIG. 2 shows a cross-section of the prepared wet collagen membrane at high magnification.

[0074] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents may occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes that fall within the true spirit of the invention.

Claims

1. A layered structure comprising at least two collagen layers having different densities.

2. the at least two collagen layers include at least one first collagen layer and at least one second collagen layer; The layered structure of claim 1 , wherein the at least one second collagen layer has a density that is up to about 90% of the density of the first collagen layer.

3. the at least two collagen layers include at least two first collagen layers and at least one second collagen layer; The layered structure of claim 1 , wherein the at least one second collagen layer is disposed between each of the at least two first collagen layers.

4. 4. The layered structure of claim 3, wherein the at least one second collagen layer each independently has a density up to about 90% of the density of the at least two first collagen layers.

5. 10. A method for preparing the layered structure of claim 1, comprising: introducing collagen into a container; fibrillating the collagen to provide a collagen gel comprising collagen fibrils; compressing said collagen gel in the presence of at least one separating element to provide at least two layers with different densities; cross-linking the collagen gel; and drying the cross-linked collagen.

6. A surgical scaffold for soft tissue growth / augmentation comprising the layered structure of any one of claims 1 to 4.

7. A method of reinforcing soft tissue comprising applying a layered structure according to any one of claims 1 to 4.

8. A method of treating bone growth / augmentation, including guided bone regeneration and guided tissue regeneration, comprising the step of applying a layered structure according to any one of claims 1 to 4.

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