Patent Application
20080081362
Some features and advantages of the invention are described with reference to the drawings of certain preferred embodiments, which are intended to illustrate and not to limit the invention.
It should be understood that this invention is not limited to the particular methodology, protocols, etc., described herein and, as such, may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.
As used herein and in the claims, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly indicates otherwise. Thus, for example, a reference to a cell may be a reference to one or more such cells, including equivalents thereof known to those skilled in the art unless the context of the reference clearly dictates otherwise. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages may mean ±1%.
All patents and other publications identified are incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
Unless defined otherwise, all technical terms used herein have the same meaning as those commonly understood to one of ordinary skill in the art to which this invention pertains. Although any known methods, devices, and materials may be used in the practice or testing of the invention, the preferred methods, devices, and materials in this regard are described here.
The present invention provides for a layered composite device and method to use the device such that the invention assists with tissue augmentation and regeneration. More specifically, multiple layers of biocompatible scaffolding and autologous tissue may provide the means to promote growth of tissues containing more than one type of cell. When utilized, these cellularized patches will be able to fill holes, repair areas of damage, or increase the surface area of tissues in hollow organs. The device may not only hold the autologous cellular and tissue in place, but may also contain the means for fixing the implant.
Recent publications have discussed various scaffolding approaches for reconstruction of skeletal or tract tissues. For example, U.S. Patent Application Pub. No. 20050154458 refers to an “active bio layer” such as hyaluronic acid, capable of interacting with stem cells from bone marrow, sandwiched between opposed surfaces of biomaterials or synthetic polymer materials, for skeletomuscular applications. The present invention does not require an “active bio layer” and is not limited to such skeletomuscular applications. U.S. Patent Application Pub. No. 20040225247 refers to a tissue patch having a protective layer for repairing an alimentary tract lesion. The present invention has no such “protective liner,” nor is it limited to such lesions. U.S. Patent Application Pub. No. 20050272153 refers to a metal coated scaffold (and thus non-degradable) with biocompatible material (but not cells or tissue) for implantation into or in place of bone. Finally, U.S. Pat. No. 6,143,293 refers to a stack of cell-seeded hydroxyapatite scaffolds for use as a 3-dimensional void filler for use in, for example, bone. The present invention is not directed to such void filler uses.
In one embodiment of the present invention, each composite contains at least two scaffolding layers and at least one cellular layer. See, for example,
The scaffold layers of the present invention are bioabsorbable, meaning that they are biocompatible and biodegradable. Biocompatible refers to materials which do not have toxic or injurious effects on biological functions. Biodegradable refers to material that can be absorbed or degraded in a patient's body. Representative materials for forming the biocompatible structure include natural or synthetic polymers, such as, for example, collagen, poly(alpha esters) such as poly(lactic acid), poly(glycolic acid), polyorthoesters and polyanhydrides and their copolymers, which degrade by hydrolysis at a controlled rate and are reabsorbed. These materials provide the maximum control of degradability, manageability, size and configuration. Other biodegradable polymer materials include polyglycolic acid and polyglactin. See, e.g., U.S. Pat. No. 5,514,181.
Other biodegradable materials include cellulose ether, cellulose, cellulosic ester, fluorinated polyethylene, phenolic, poly-4-methylpentene, polyacrylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate, polycyanoarylether, polyester, polyestercarbonate, polyether, polyetheretherketone, polyetherimide, polyetherketone, polyethersulfone, polyethylene, polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide, polysulfone, polytetrafluoroethylene, polythioether, polytriazole, polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose, silicone, urea-formaldehyde, or copolymers or physical blends of these materials.
An example of a biocompatible, biodegradable polymer suitable for the instant invention is polyglactin, manufactured as Vicryl® (Novartis-Ethicon). Vicryl® (polyglactin 910) is a 90:10 copolymer of glycolide and lactide, derived respectively from glycolic and lactic acids.
Regarding the use of ceramics as material for scaffold formation, a bioactive glass such as the commercially available BioGlass® (NovaBone Products, LLC, Alachua Fla.), can be modified with a poly(lactic co-glycolic acid) polymer matrix. See, e.g., U.S. Pat. No. 6,328,990. “Bioactive” means that the material has the ability to interact or bind to living tissue.
Alternatively, hydrogels such as alginate-RGD may be used. Alginates are seaweed-derived copolymers for which the rigidity of the hydrogel may be controlled by crosslinking its glucuronate residues with, e.g., calcium or adipic dihydrazide. Alginate may further be modified with cell-adhesive peptides such as Arg-Gly-Asp (RDG) peptide to promote cellular attachment to the scaffold layer. See, e.g., Wong et al., 570 Science 119-33 (2004); Das & Hollister, Tissue Engineering Scaffolds in Encyclopedia of Mats: Sci & Tech. 1-7 (Elsevier Sci., Ltd. 2003).
Further regarding the scaffold layer, an optional pharmaceutical or bioactive agent may be incorporated into the scaffolding. The variety of different pharmaceuticals that can be used in conjunction with the scaffolds of the present invention is vast. Such pharmaceuticals or agents will in general be selected according to the tissue or organ being reconstructed or augmented, to ensure that appropriate new tissue is formed in the engrafted organ or tissue (for examples of such additives for use in promoting bone healing, see, e.g., Kirker-Head, 24(5) C. A. Vet. Surg. 408-19 (1995)). Common pharmaceuticals and bioactive agents which may be administered via the pharmaceutical compositions of the invention include, without limitation: anti-infectives such as antibiotics and antiviral agents; chemotherapeutic agents; anti-rejection agents; analgesics and analgesic combinations; anti-inflammatory agents; hormones such as steroids; growth factors; and other naturally derived or genetically engineered proteins, polysaccharides, glycoproteins, or lipoproteins.
Scaffolds containing these materials may be formulated by mixing one or more agents with the material used to make the scaffold. Alternatively, an agent could be coated onto the scaffold, preferably with a pharmaceutically acceptable carrier. Any pharmaceutical carrier can be used that does not dissolve or react with the scaffold. The pharmaceutical agents may be present as a liquid, a finely divided solid, or any other appropriate physical form. Typically, but optionally, they will include one or more additives, such as diluents, carriers, excipients, stabilizers or the like. Additionally, such optional agent or bioactive agent may be added separately (i.e., not manufactured into the scaffold matrix. For example, a bioactive agent such as fibrin may be added to the tissue or cell layer, and may serve as a bioactive glue between the cell or tissue layer and the scaffold layer(s).
An aspect of the present invention provides for a composite comprising distinct scaffold layers having different physiochemical properties. For example, it is known that chemical, topographical, and mechanical cues affect cellular responses at the cell-biomaterial interface. See Wong et al., (2004). Moreover, considerations of mechanical strength in maintaining rigidity relating to a particular organ's structure and placement may suggest using a particular scaffold layer in that context. Hence, for example, a composite according to the present invention may comprise a rigid scaffold layer that provides mechanical strength in one layer, and second scaffold layer that promotes cellular ingrowth. Alternatively, different layers might be used to promote the growth of distinct cell types found in complex tissues. For example, one scaffold layer might promote the growth of cartilage, while a second scaffold layer promotes the growth of bone. Or, for example, one scaffold layer might have porosity that will foster muscle-cell infiltration while another layer might have porosity that would exclude larger cells and allow only smaller cell infiltration. Alternatively, the different scaffold layers may comprise polymers that degrade at different rates such that one layer degrades before the other. For example, gamma-irradiated PLGA degrades faster and might be used in one layer (e.g., on the inner surface of a hollow organ), while polyethylene oxide degrades more slowly and might be used in another layer (e.g., on the exterior surface of a hollow organ).
The living component of the present invention may be autologous cells or autologous tissue, obtained by any number of techniques well-known in the art. For example, during surgery a tissue sample may be obtained and simply placed on a scaffolding layer. Alternatively, tissues containing more than one cell type may be separated, for example with a scalpel, into substantially distinct tissue samples. One or more of the separated tissue samples may then be used with the scaffolding, or the separated tissues may be celularized before placement on the scaffold. Such cellularization techniques, such as mincing or treating with appropriate cellularizing agents, are known in the art.
Alternatively, the tissue or cellularized (cell) sample may be treated in vitro before being placed on the scaffold layer. For example, cells (such as autologous cells) can be cultured in vitro to increase the number of cells available for seeding on the scaffold(s). The use of allogenic cells, and more preferably autologous cells, is preferred to prevent tissue rejection. In certain embodiments, chimeric cells, or cells from a transgenic animal, can be seeded onto the polymeric matrix. Cells can also be transfected prior to seeding with genetic material. Useful genetic material may be, for example, genetic sequences which are capable of reducing or eliminating an immune response in the host. For example, the expression of cell surface antigens such as class I and class II histocompatibility antigens may be suppressed. This may allow the transplanted cells to have reduced chance of rejection by the host. In addition, transfection could also be used for gene delivery. Urothelial and muscle cells could be transfected with specific genes prior to polymer seeding. The cell-polymer construct could carry genetic information required for the long term survival of the host or the tissue engineered neo-organ.
The composite of the present invention may be useful in treating organs. In particular, hollow organs, such as bladder, urethra, jejunum, esophagus, trachea, colon, and stomach may benefit from placement of the present composite as a “patch” in an area requiring tissue augmentation or regeneration. For example, regarding the bladder, if an area of the bladder is missing due to congenital defect or has been lost due to disease, injury or surgery (e.g., partial cystectomy), the patient may benefit from having the bladder area increased or restored to the original size as the particulars of the case allows.
In an aspect of the present invention, sheets of scaffold materials are provided in a sterile form such that the physician, or other member of the surgical team, may cut the size of the particular scaffold to a size as required by the instance at hand. Multiple types of scaffold with desired physiochemical properties (as discussed above) may be provided in the same or in different packages.
An embodiment of the present invention allows for placement of the composite in a hollow organ such that one exterior scaffold layer may be seated upon the outside surface of the organ and the opposite exterior scaffold layer may be seated upon the inside of the organ. In such arrangement, the interior composite layers, comprising at least one tissue or cell layer (and optionally additional tissue or cell layer(s) that may or may not be further separated by additional scaffold layer(s)), to be aligned with and adjacent to the hollow organ tissue layer. So, for example, regarding the bladder, one exterior scaffold layer would rest on the serosal layer (tunica seros), and the opposite exterior scaffold layer would rest on the urotheliuem. Tissue layers might include, for example, detrusor (tunica muscularis) and lamina propria, each tissue layer positioned within the composite such that they may be aligned with the native organ tissue upon implantation. The composite is then fixed in place with, for example, suture. Such placement facilitates vascularization and cell organization as the composite integrates into the organ.
While reference is made herein to augmentation of bladder according to the invention, it will be understood that the methods and materials of the invention are useful for tissue reconstruction or augmentation of a variety of tissues and organs in a subject. Thus, for example, organs or tissues such as bladder, ureter, urethra, renal pelvis, and the like, can be augmented or repaired with polymeric scaffolds seeded with cells. The materials and methods of the invention further can be applied to the reconstruction or augmentation of vascular tissue (see, e.g., Zdrahala, 10(4) J Biomater. Appl. 309-29 (1996)), intestinal tissues, stomach (see, e.g., Laurencin et al., 30(2) J Biomed Mater. Res. 133-38 (1996)), and the like. The patient to be treated may be of any species of mammals such as a dog, cat, pig, horse, cow, or human, in need of reconstruction, repair, or augmentation of a tissue.
In one embodiment of the invention, living cells are sandwiched between the scaffolding layers and substantially cover the surface area of the scaffold material. See, for example,
In yet another embodiment of the invention, the dissected tissue is left in its natural state and fixed between scaffold layers. See, for example,
Another embodiment of the invention provides for a method of treating hollow organ tissue using the patch of the present invention. For example, as shown in
Urothelium cells and smooth muscle cells (SMC) were isolated from porcine bladder and cultured in a humidified incubator at 37° C., 5% carbon dioxide and 95% air for one week until the cells reached 85% confluency. Porcine bladder smooth muscle cells were statically seeded at a density of 2×106 cells/scaffold onto 90/10 PGA/PLA 11×7 mm scaffold discs. The SIS scaffolds were similarly prepared and seeded with porcine bladder urothelium cells at a density of 2×106 cells/scaffold. The scaffolds were incubated in a humidified incubator at 37° C. for 2 hours, after which the 90/10 PGA/PLA and SIS scaffolds were sutured together with 4-0 VICRYL coated suture (ETHICON) with both the cell-seeded surfaces placed internally and in contact with each other. The cell-seeded scaffolds were then cultured with 50% Keratinocyte-SFM medium (Invitrogen Co) and 50% SMC medium (Cambrex). After 2 weeks, the scaffolds were evaluated by histology (H&E, alpha smooth muscle actin stain, Cytokeratin-7). Both the urothelium and smooth muscle cells were retained in their respective scaffolds and retained their phenotypes as evidenced by immunostaining.
Coated 90/10 PGA/PLA scaffolds were prepared by dipping the scaffolds in a 5% solution of 50/50 PGA/PLA to increase the stiffness of the scaffolds. Urothelium cells and smooth muscle cells were isolated from porcine bladder and cultured as described in example 1. Urothelium cells were loaded onto the coated 90/10 PGA/PLA scaffolds with a cell density of 2×106 cells/scaffold. Uncoated 90/10 PGA/PLA nonwoven scaffolds were seeded with porcine bladder smooth muscle cells. The cell-seeded scaffolds were incubated as in example 1 and after 2 hours incubation the cell seeded scaffolds were sutured together and cultured as described in example 1. After two weeks the scaffolds were evaluated by histology (H&E, alpha smooth muscle actin stain, Cytokeratin-7). Both the urothelium and smooth muscle cells were retained in their respective scaffolds and retained their phenotypes as evidenced by immunostaining.
A small 2 cm-by-2 cm piece of tissue is excised from a normal healthy bladder. The smooth muscle cell layer is then removed from the urothelial cell layer using a scalpel. This creates two distinct tissue samples for mincing. Each sample is then processed under sterile conditions to create a suspension having at least one minced, or finely divided, tissue particle.
The particle size and shape of each tissue fragment may vary. For example, the tissue size can range from about 0.1 mm3 and 3 mm3, or in the range of 0.5 mm3 and 1 mm3, or in the range of 2 mm3 and 3 mm3, or less than about 1 mm3. The shape of the tissue fragments can include, for example, slivers, strips, flakes, or cubes.
Each sample of tissue is subsequently spread on a separate 3 cm-by-3 cm square Polyglactin 910 scaffold (300 mg/cc, 1 mm thick), leaving bare ½ cm around the scaffold perimeter. This results in two scaffolds with two different tissue types. Fibrin glue is spread on a single scaffold and the two scaffolds are sandwiched into a five-layer composite (scaffold, minced tissue A, fibrin, minced tissue type B, scaffold).
Larger cuts are made in the patient's bladder in a shape that eases the placement of the implant. Each scaffold is fixed by passing sutures through a layer of scaffold, the native tissue layer, and then next layer of scaffold. In this way, the scaffold is situated to sandwich the native tissue and also minced tissue. The interfaces of the tissue will also match up so that vascularization and cell organization is facilitated.
A small 2 cm-by-2 cm piece of tissue is excised from a normal healthy bladder. The smooth muscle cell layer is then removed from the Urothelial cell layer using a scalpel. This creates two distinct tissue samples having different cell types. Each sample is put through a digestion process to isolate individual cells. Once isolated, the cells are suspended in a collagen gel.
Each cell-collagen suspension is subsequently spread on a separate 3 cm-by-3 cm square Polyglactin 910 scaffold (300 mg/cc, 1 mm thick), leaving bare ½ cm around the scaffold perimeter. This results in two scaffolds with two different tissue types. The two scaffolds are sandwiched together to form a four-layer composite (scaffold, cell-collagen suspension A, cell-collagen suspension B, scaffold).
Larger cuts are made in the patient's bladder in a shape that eases the placement of the implant. Each scaffold is fixed by passing sutures through a layer of scaffold, the native tissue layer, and then next layer of scaffold. In this way, the scaffold is situated to sandwich the native tissue and also minced tissue. The interfaces of the tissue will also match up so that vascularization and cell organization is facilitated.
A small 2 cm-by-2 cm piece of tissue is excised from a normal healthy bladder. Small cuts are made in the tissue at various intervals, and the tissue then stretched into a 3 cm-by-3 cm sample, the stretching creating voids where the cuts have been made. Biocompatible filler such as fibrin glue, may be placed inside the void to maintain sample shape and facilitate healing.
The processed sample is then sandwiched between a 4 cm-by-4 cm square Polyglactin 910 scaffold (300 mg/cc, 1 mm thick), leaving bare ½ cm around the scaffold perimeter. The construction creates a 3-layered composite (scaffold, processed tissue, scaffold).
Larger cuts are made in the patient's bladder in a shape that eases the placement of the implant. Each scaffold is fixed by passing sutures through a layer of scaffold, the native tissue layer, and then next layer of scaffold. In this way, the scaffold is situated to sandwich the native tissue and also minced tissue. The interfaces of the tissue will also match up so that vascularization and cell organization is facilitated.
Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
| Number | Date | Country | |
|---|---|---|---|
| 60847922 | Sep 2006 | US |
