Aligned scaffolds for improved myocardial regeneration
http://www.pharmcast.com/Patents200/Yr2008/June200 [2008-6-27]
Tag : l-lysine acetate
Abstract
The present invention relates to a biocompatible, three-dimensionalscaffold useful to grow cells and to regenerate or repair tissue inpredetermined orientations. The scaffold is particularly useful forregeneration and repair of cardiac tissue. The scaffold containslayers of alternating A-strips and S-strips, wherein the A-stripswithin each layer are aligned parallel to each other andpreferentially promote cellular attachment over attachment to theS-strips. Methods of producing and implanting the scaffold are alsoprovided.
Description of the Invention
SUMMARY OF THE INVENTION
The present invention is directed to a semi-solid,three-dimensional, biocompatible scaffold for cell growth, tissuerepair or regeneration, comprising one or more layers ofalternating attachment-strips ("A-strips") andseparating-strips ("S-strips") wherein the A-stripswithin each layer are aligned parallel to each other andpreferentially promote cellular attachment over attachment to theS-strips. The present invention is also directed to methods ofproducing and implanting such scaffolds.
In one aspect, the scaffold is biodegradable and re-shapeable.After implantation of the scaffold into an area of tissue damage ordisease, cells migrate into the scaffold and preferentially attachto the A-strips allowing cells to align in accordance with thearchitecture of the scaffold. The architecture of the scaffold canthus provide spatial- and orientation-dependent signals for cellgrowth and/or differentiation. As the cells grow and differentiate,they reshape the architecture of the scaffold such that thescaffold is adopted into the surrounding tissue. Eventually thesescaffolds completely degrade leaving behind the integrated cellulartissue and tissue-specific extracellular matrix.
The design of the scaffolds can be varied in relation to differenttissue architectures by varying the dimensions of the elements ofthe scaffolds, and/or the materials that the elements of thescaffold are made of. The elements of the scaffold are theA-strips, S-strips and the separating layers, and their dimensionsas to width, length, thickness and porosity are fixed according toa scaffold's intended use in the repair, regeneration or growth ofa particular tissue or cell type, in vivo or in vitro. The width ofthe A-strips and S-strips are fixed to promote the orderedalignment of cells within a layer so that cells can receivespatial- and orientation-dependent signals for cell growth and/ordifferentiation from each other and from the strip materials. Thematerials of the scaffold are also chosen such that the A-stripspromote the adhesion of cells more than the S-strips and theseparating layers, and such that the porosity of the S-strips andseparating layers allow a homogeneous distribution of cellsthroughout the scaffold. Further, the scaffold can be supplementedor treated with bioactive agents such as those, for example, thatpromote cell migration and proliferation.
In accordance with the invention, the scaffold can be implanted ina subject at a site of tissue damage without having been pre-seededwith cells. In this embodiment, the scaffold is seeded with cellsin situ either by endogenous cells or by cells separately implantedinto the subject. Alternatively, the scaffold can be pre-seededwith cells in vitro prior to implantation in a subject.
In another aspect of the present invention, methods of producingthe scaffolds of the inventions are provided. In one embodiment, amethod of producing a semi-solid, three-dimensional, biocompatiblescaffold comprises layering one or more sheets of biocompatiblestrip material by a fixed distance from each other; cutting thesheets to provide A-strips of a fixed width, maintaining theparallel A-strips at a fixed distance from each other; andcontacting the parallel A-strips with a biocompatible material thatdoes not promote cellular attachment under conditions to fill thespace separating the A-strips, thereby forming S-strips, and tofill the space separating the layers; to form a semi-solid,three-dimensional, biocompatible and, preferably, biodegradablescaffold.
DETAILED DESCRIPTION OF THE INVENTION
The issued U.S. patents, published and allowed applications, andreferences cited herein are hereby incorporated by reference.
The present invention relates to a biocompatible, three-dimensionalscaffold useful for growing cells, regenerating or repairingtissue. This invention also provides methods of producing andimplanting the scaffold. The semi-solid, three-dimensional,biocompatible scaffold for cell growth, tissue repair orregeneration, or any combination thereof, comprises one or morelayers of alternating attachment-strips ("A-strips") andseparating-strips ("S-strips"), wherein the A-stripswithin each layer are aligned parallel to each other, and whereinthe A-strips preferentially promote cellular attachment."Attachment-strips" or "A-strips" are definedherein as an element of the scaffold that preferentially promotescellular attachment over other elements of the scaffold."Separating-strips" or "S-strips" are definedherein as an element of the scaffold that does not preferentiallypromote cellular attachment, and in relation to the architecture ofthe scaffold, serves to separate the A-strips from each otherwithin a scaffold layer.
The dimensions and the materials of the scaffold are designed toallow cells to align in a manner to receive spatial- and/ororientation-dependent cues or signals from other cells and/or fromthe materials that constitute the A-strips of the scaffold. Thesesignals or cues help the cells in the scaffold to grow anddifferentiate so that the cells can adopt to and/or integrate intothe surrounding tissue in which the scaffold is implanted.Additionally, the scaffold can also provide the cues or signalssuch that cells in the scaffold grow and differentiate intofunctional tissues in vitro.
A. Structure and Materials of the Scaffold
The scaffold of the present invention has from one to many layers.Preferably the scaffold comprises multiple alternating layers ofscaffold layers and separating layers. The scaffold layer iscomposed of alternating A-strips and S-strips where the A-stripswithin the scaffold layer are aligned parallel to each other. Inaccordance with the invention, A-strips preferentially promote theattachment of cells, and S-strips and separating layers do notpreferentially promote the attachment of cells. As used herein,"preferentially promotes" means that more cells attach tothe A-strips rather than to the S-strips or the separating layers.In addition, preferentially promoting attachment can be achieved bypreferential proliferation or differentiation of the cells on theA-strips rather than on the S-strips or the separating layers.
It is preferred that greater than about 70% of the cells within ascaffold are present on A-strips. It is more preferred that greaterthan about 90% of the cells within a scaffold are present onA-strips. The percentage of cells present on A-strips as comparedto S-strips can be tested by performing a 2-dimensional cellculture experiment where the culture surface is coated with S-stripand A-strip material in an area and design similar to one layer ofa desired scaffold. A cell type will be used which is relevant tothe intended target tissue of the scaffold.
The A-strips can range from about 20 micrometers to about 200micrometers in thickness, and from about 20 micrometers to about100 micrometers in width. These dimensions, especially the width,are relevant to the cell-attachment properties of the A-strip. Forexample, if the A-strips are too narrow (i.e., if the A-strips aremore narrow than the size of a cell), then cells cannot properlyattach. If the A-strips are too wide, then cells may not properlyor efficiently align in relation to the architecture of the layerbecause the cells cannot align on the A-strips in a linear fashion.For use with cardiomyocytes, the preferred width of the A-strips isfrom about 20 to about 50 micrometers in width.
The S-strips can range from about 20 micrometers to about 200micrometers in thickness, and from about 20 micrometers to about200 micrometers in width. If the S-strips are too narrow, i.e.,similar to the width of a single cell, then the A-strips cannotfunction to align the cells in a parallel orientation. For example,if the cells on one A-strip are able to span the distance of theS-strip to another A-strip, then the A-strips are less effective inaligning cells in a substantially linear fashion on an A-strip. Forcardiomyocytes, the preferred width of an S-strip is from about 50to about 100 micrometers in width.
The separating layer alternates with the scaffold layer. As definedherein, a "scaffold layer" is a layer of a scaffoldcomprising alternating A-strips and S-strips. In other words, theseparating layer serves to separate the scaffold layers from eachother. According to the present invention, a separating layer canbe made of a material suitable for a S-strip. Hence, a separatinglayer is made of a material that does not preferentially promotecell attachment and/or proliferation and/or differentiation. Thus,the A-strips of the scaffold preferentially promote cell attachmentover the S-strips and the separating layers.
The separating layer can range from about 20 micrometers to about200 micrometers in thickness. Further, the separating layer and theS-strips can, but need not necessarily contain pores. The poresallow cell migration and can range from about 10 micrometers toabout 300 micrometers in diameter. The pore size cannot exceed thedimensions of a layer. The pores allow cells to migrate throughoutthe scaffold, to aid in providing an even or homogeneousdistribution of cells throughout the scaffold. The pores alsoenable the migration of soluble peptides and proteins that arenecessary for cellular growth and communication. The pores alsoenable efficient vascularization between the scaffold and thesurrounding tissue.
The alignment of the A-strips within a scaffold layer is parallel.However, when the scaffold is multi-layered, the alignment of theA-strips between successive scaffold layers need not be parallel.For example, the scaffold can be constructed such that there is nooverall alignment of the A-strips from scaffold layer to scaffoldlayer. This overall random alignment serves to grow and regeneratetissue where cell alignment is not ordered, for example, in skin.Of course, the scaffold can be constructed such that all theA-strips are aligned in one general parallel orientation, or in anyother desired pattern, e.g., such as alternating 90.degree. angles.
The scaffold is preferably composed of a material that permitscells to reshape the scaffold. Such properties allow the cells ofthe scaffold to become integrated with or adopted into thesurrounding tissue in which the scaffold is implanted. By"integrating" or "adopting", the cells of thescaffold are not rejected by the surrounding tissue in which thescaffold is implanted. The scaffold is more re-shapeable when thematerials of the scaffold are biodegradable and not synthetic.
The A-strips of the present invention are constructed from abiocompatible substance that can be formed into a shape thatessentially resembles a strip or a fiber having the dimensionsstated herein. Biocompatible substances that can be used to formthe A-strips, include but are not limited to, extracellular matrixmaterial, proteins or peptides that are not present inextracellular matrices but have cell attachment properties, andsynthetic materials, provided such materials can be formed intoA-strips of the appropriate size and have the requisite biologicalcharacteristics to preferentially promote cell attachment and/orproliferation and/or differentiation. Non-synthetic, biocompatibleand biodegradable materials are preferred for A-strips, such asextracellular matrix material. If synthetic materials are used asstrip material, the synthetic materials can be combined with eitherextracellular matrix material or proteins or peptides withcell-attachment properties so that the strips preferentiallypromote cell attachment or proliferation.
According to the present invention, "extracellular matrixmaterial" is any material or substance that is present in anextracellular matrix. An extracellular matrix is an acellular sheetor layer that consists of three major classes of biomolecules: (1)structural proteins such as collagen and elastin, (2) specializedproteins such as, perlacan, agrin, laminin, fibronectin, entactin,nidogen, and fibrillin, and (3) proteoglycans which are composed ofa protein core to which is attached long chains of repeatingdisaccharide units (glycosaminoglycans (GAGs)). Thus, according tothe present invention, any of the above-types of biomolecules,individually or in combination, in natural, processed orrecombinant forms, are considered extracellular matrix material.
The extracellular matrix material can be isolated from animals orfrom an in vitro cellular source rich in producing extracellularmatrix material. Sheets of extracellular matrix isolated fromanimals can be cut to form the A-strips of the scaffold.Alternatively, sheets of isolated extracellular matrix material canbe processed into gels or liquids (see for example, U.S. Pat. No.5,275,826 or U.S. Pat. No. 4,829,000), and these gels or liquidscan be molded to form the A-strips of the scaffold. Additionally,recombinant forms of extracellular matrix materials can be used toform the strips of the scaffold.
The extracellular matrix material can be isolated from a variety oftissue sources, for example, essentially any submucosa. A submucosais a layer of areolar connective tissue that lies beneath themucosa. Examples of submucosa that can be used as a source ofextracellular matrix material, include intestine submucosa, stomachsubmucosa, liver submucosa, and urinary bladder submucosa. Basementmembranes can also be used as a source of extracellular matrixmaterial. Basement membranes are thin, but continuous sheets thatseparate epithelium from stroma and surround nerves, muscle fibers,smooth muscle cells and fat cells. Additionally, tissues such asbone, bone marrow, cartilage and placenta can also be sources ofextracellular matrix material. Further, embryonic and fetal cardiactissue can be sources of extracellular matrix material.Representative processes that can be used to isolate extracellularmatrix material from tissue are described in, Voytik-Harbin, S. L.,"Three-dimensional extracellular matrix substrates for cellculture," Methods Cell Biol., (2001), 63:561-81.
The tissue sources of extracellular matrix material can be isolatedfrom any mammal, including but not limited to: pig, human, cow,sheep, goat, donkey, horse, rabbit, dog, cat, rat and mouse. Theextracellular matrix material can be in the form of a sheet fromwhich A-strips are cut, or the material can be processed into theirseparate components or combinations of components, and then formedinto A-strips or a sheet. Such sheets can then be cut into A-stripsin accordance with the invention. Alternatively, if individualA-strips are formed, these A-strips have the dimensions as statedherein and are arrangeable in a parallel orientation.
Sheets of extracellular matrix can be isolated from essentially anytype of submucosa. For example, in U.S. Pat. No. 4,902,508, bydelaminating the tunica muscularis and at least the luminal portionof the tunica mucosa of the small intestine, a tunica submucosa wasisolated. As described in U.S. Pat. No. 6,099,567, the stomachsubmucosa can be isolated by delaminating the smooth muscle layersof the muscularis externa and at least the luminal portion of themucosal layer of a segment of the stomach. As described in U.S.Pat. No. 5,554,389, urinary bladder submucosa is isolated bydelaminating the abluminal muscle cell layers and at least theluminal portion of the mucosal layer of a segment of urinarybladder. Acellular and detoxified sheets of submucosa arecommercially available, for example, see COOK.RTM. BiotechIncorporated, SIS.TM. Technology (COOK Biotech Incorporated, 3055Kent Avenue, West Lafayette, Ind. 47906 USA).
Extracellular matrix material can also be isolated as a slurry orsolution. Solutions or slurries can be processed or molded intoA-strips of the appropriate dimensions, for example, by thefollowing processes: freeze-drying in a mold; air drying intoA-strips; air drying into sheets which are cut into A-strips;gelled by neutralizing pH; and solutions or slurries can becombined with a second material (e.g., collagen, gelatin,self-assembling peptides) which can be solidified, cross-linked, orotherwise turned into a solid without affecting the biologicalactivity of the extracellular matrix material.
Processed or recombinant forms of extracellular matrix materialscan be used as A-strip material. For example, U.S. Pat. No.4,829,000 reports a processed, reconstituted,basement-membrane-derived extracellular matrix composition(MATRIGEL.TM.). Also, U.S. Pat. No. 5,275,826 reports fluidizedforms of submucosa. Purified or recombinant forms of collagen,elastin, perlacan, agrin, laminin, fibronectin, entactin, nidogen,fibrillin, proteoglycans can be used as strip material, and arecommercially available. For example, BD BIOSCIENCES.TM. offers avariety of ECM products that can be used in the present invention:BD Matrigel.TM. Basement Membrane Matrix, Collagen I ECM, CollagenIII ECM, Collagen IV ECM, Collagen V ECM, Fibronectin ECM, LamininECM.
The extracellular matrix material can be from a syngeneic,allogeneic or xenogeneic source. When used as the materialcomprising the A-strips of the scaffold, the extracellular matrixmaterial is acellular, and should not be recognized as"foreign" by a host's immune system, and therefore shouldnot be rejected by the host. Further, the biocompatible materialsthat comprise the scaffold are substantially endotoxin free.
Proteins and peptides that have cell-attachment properties but arenot present in extracellular matrices can also be used as A-stripmaterial. Such proteins and peptides include, but are not limitedto, vitronectin, polypeptides with the amino acid sequencearginine-glycine-aspartic acid (`RGD sequence`), and poly-L-lysine.According to the present invention, proteins and peptides that havecell attachment properties are proteins or peptides that bind tomolecules on a cell surface with an affinity such that the bindingis not transient.
Synthetic materials such as polylactic acid (PLA), poly-glycolicacid (PGA), co-poly-lactic/poly-glycolic acid polymers (PLGA), canalso be used as A-strip material, if a non-biodegradable scaffoldis desired. If synthetic materials are used as A-strip material,the synthetic materials can be combined with either extracellularmatrix material or proteins or peptides with cell-attachmentproperties so that the A-strips preferentially promote cellattachment. Preferably, the A-strips of the present invention aremade from extracellular matrix material or proteins or peptideswith cell attachment properties.
The S-strips and the separating layers of the invention arebiocompatible and preferably biodegradable substances that do notpreferentially promote cell attachment. Preferably, the S-stripsand the separating layers do not permit cells to substantiallyattach. The material of the S-strips and the material of theseparating layer do not have to be identical. Examples of S-stripsand separating layer material include, but are not limited to,alginate, agarose, polylactic acid (PLA), poly-glycolic acid (PGA),co-poly-lactic/poly-glycolic acid polymers (PLGA), gelatin,ethylene-vinyl acetate, fibrin, sucrose octasulfate, dextran,polyethylene glycol, polyacrlyamide, cellulose, latex,polyhydroxyethylmethacrylate, nylon, Dacron,polytetrafluoro-ethylene, polystyrene, polyvinylchlorideco-polymer, cat gut, cotton, linen, polyester, and silk. Alginate,agarose and gelatin, are preferred. It is also preferred that allof the elements of the scaffold have a similar rate ofbiodegradation.
When the S-strips and separating layers comprise alginate, theporosity of alginate can be manipulated by using different sourcesof alginate, by varying the concentration of alginate, and byvarying the divalent cations used to help polymerize the alginate.
Proteins or peptides can be micro-patterned on the surface ofbiocompatible sheets, A-strips, S-strips or separating layers. Inthe present invention, proteins or peptides are `micro-patterned`by immobilizing proteins or peptides in micrometer sized shapes onthe surface of biocompatible materials.
Advances in patterning technology have generated a range oftechniques with which biomolecules can be immobilized on surfaceswith microscale precision. Some techniques that can be used withthe present invention include microcontact printing,photolithography, photochemistry, 3D printing, and microwriting.These techniques are known to those of skill in the art.
The scaffold of the present invention can also be treated withbioactive agents. These agents can be used alone or in combinationand include, but are not limited to, vascularization-promotingfactor, a cytokine, a growth factor, an enzyme, a hormone, anangiogenesis factor, a vaccine antigen, an antibody, a clottingfactor, a regulatory protein, a transcription factor, a receptor, astructural protein, and any functional fragment, variant orcombinations thereof. Preferably, the agents are located on or inthe A-strips of the scaffold, although they can be present in theS-strips and the separating layers, provided they do not alter theoverall characteristic of the scaffold to promote cellularattachment to the A-strips. Agents that recruit cells or directcells include cytokines, growth factors, enzymes, hormones,angiogenesis factors, regulatory proteins, transcription factors,receptors, structural proteins and any of their bioactivefunctional fragments. Agents that promote cell attachment includereceptors and structural proteins. Agents that promotevascularization of the scaffold and tissue include vascularizationpromoting factors, cytokines, growth factors and angiogenesisfactors.
Examples of specific bioactive agents include, but are not limitedto, collagen, laminin, fibronectin, granulocyte-colony stimulatingfactors (G-CSF), granulocyte-macrophage colony stimulating factor(GM-CSF), stem cell factor (SCF), vascular endothelial growthfactor (VEGF), platelet-derived growth factor (PDGF), transforminggrowth factors, human growth hormone (hGH), Factor VIII, Factor IX,erthropoietin (EPO), albumin, heme oxygenase, hemoglobin, alpha-1antitrypsin, calcitonin, glucocerebrosidase, low densitylipoprotein (LDL) receptor, IL-2 receptor, globins,immunoglobulins, catalytic antibodies, interleukins, chemokines,insulin, insulin-like growth factor 1 (IGF-1), insulinotropin,parathyroid hormone (PTH), leptin, an interferon, nerve growthfactors, epidermal growth factor (EGF), endothelial cell growthfactor, endothelial cell stimulating angiogenesis factor (ESAF),angiogenin, tissue plasminogen activator (t-PA), folliclestimulating hormone (FSH), Flt-3 ligand, megakaryocyte growth anddevelopment factor (MGDF), and 3-hydroxy-3-methyl glutaryl coenzymeA (HMG CoA) reductase inhibitors, and any functional fragment,variant or combinations thereof.
The architecture of the scaffold helps in promoting cellular growthand differentiation. Most cells can grow in vitro without anyscaffolding. Yet, many cells do not fully differentiate withoutspatially defined cell-contact and orientation signals. Forexample, integrin receptors on the surface of cells bind to RGD(arginine-glycine-aspartic acid) sequences in extracellular matrixproteins such as fibronectin. This interaction induces cellspreading and intracellular signaling. Spatial orientation helps toprovide proper cell-to-cell signaling and cellular function. Forexample, cardiomyocytes are organized into parallel cardiac musclefibers with intracellular contractile myofibrils oriented parallelto the long axis of each cell and junctional complexes betweenabutting cells concentrated at the ends of each cardiomyocyte.Without this highly oriented architecture, electromechanicalcoupling of cardiomyoctes does not occur, and the transmission ofdirected contraction over long distances is not possible. Thus,although cardiomyocytes can be grown in in vitro cultures, thecultured cardiomyocytes only spread to form an epithelioid sheet,with disorganized myofibrils and diffuse intercellular junctions.
B. Cellular Growth, Tissue Repair and Regeneration
The scaffolds of the present invention are used for both in vitroand in vivo cellular growth and differentiation. The purpose of invivo use is for the repair and regeneration of damaged and/ordiseased tissue. The scaffolds can be used as self-seedingscaffolds for cellular attachment, growth and repair in situ; orthe scaffold can be seeded in vitro with cells prior toimplantation. Generally, cells at a concentration of1.times.10.sup.6 to 5.times.10.sup.6 cells/ml are added to a dishor bioreactor containing a scaffold(s), and the cells are incubatedfor 24-72 hours at 37.degree. C. at 5% CO.sub.2 for initialseeding. However, the cell concentration can range from about1.times.10.sup.4 to about 1.times.10.sup.7, or higher for cellsthat can achieve such concentrations.
As used herein, the term "damaged or diseased tissue"means tissue in which cells have been lost or have died due toinsufficient blood supply, mechanical injury, infection,irradiation, trauma, disease, or other insult. For example, damagedand or diseased tissue includes, but is not limited to, scartissue, and tissue that is torn, crushed, or has undergone necrosisresulting from blood loss. By "necrosis" is meantpathologic cell death following irreversible damage to the cell.The damaged or diseased tissue can be distinguished from thesurrounding tissue, for example, by physical inconsistency ordiscontinuity.
Cells which can used to seed the scaffold in vitro include, but arenot limited to, side population (SP) adult stem cells, Lineagenegative (Lin.sup.-) stem cells, Lin.sup.-CD34.sup.- stem cells,Lin.sup.-CD34.sup.+ stem cells, Lin.sup.-cKit.sup.+ stem cells,mesenchymal stem cells (MSC), cord blood cells, tissue stem cells,whole bone marrow, bone marrow mononuclear cells (BM-MNCs), cardiacstem cells, tissue stem cells, endothelial progenitor cells (EPCs),skeletal myoblasts (satellite cells), muscle derived cells (MDCs),go cells, endothelial cells, adult cardiomyocytes, fibroblasts,smooth muscle cells, genetically modified cells, MyoD scarfibroblasts, pacing cells, embryonic stem cell clones, embryonicstem cells, fetal or neonatal cells, teratoma cells, and anycombination thereof.
SP cells can be isolated by a fluorescence-activated cell sorter(FACS) technique utilizing the ability of SP cells to excludeHoechst dye from the nucleus. In addition to bone marrow, SP cellshave been isolated from most tissues, including cardiac andskeletal muscle. By the more common surface identification, thesecells are Lin.sup.-, Sca-1.sup.+, c-Kit.sup.+, CD43.sup.+'CD45.sup.+' CD34.sup.-.
Bone marrow cells are isolated, and all the cells that havedifferentiated to a specific lineage are removed, either by FACSsorting or magnetic-bead sorting. The cells that remain are thestem and progenitor cells, or Lin.sup.- cells. Of these cells, themost primitive bone marrow-derived stem cells areLin.sup.-CD34.sup.-. Cells that are Lin.sup.-CD34.sup.+ includehematopoietic stem cells. Lin.sup.-cKit.sup.+ cells express cKit,which is the cell surface receptor for stem cell factor. Therefore,Lin.sup.-cKit.sup.+ cells are often used as a stem cell population.Lin.sup.-cKit.sup.+ cells have been isolated from the heart and thebone marrow.
Whole bone marrow can be used for transplantation and scaffoldseeding. The whole bone marrow sample is filtered to remove boneparticles. Whole bone marrow includes many extracellular matrixproteins and growth factors. The BM-MNC population can be separatedfrom whole bone marrow by a density gradient centrifugationprocedure. The BM-MNC population contains non-granular white bloodcells, progenitor cells, and stem cells. Although most work to datehas focused on isolating stem cells from the bone marrow, some stemcells (e.g., SP, Lin.sup.-cKit.sup.+) can be isolated from tissues,for example fat and cardiac muscle.
MSCs are stem cells that ordinarily differentiate into the cells ofmesenchymal tissues (e.g. bone, cartilage, fat), but can alsodifferentiate into cardiomyocytes under certain conditions. MSCsare isolated from the bone marrow, and have the capacity toproliferate in vitro. EPCs are also isolated from the bone marrowbased on cell surface markers. EPCs differentiate into endothelialcells, and when transplanted to ischemic tissue, these cells canform new blood vessels.
Cord blood cells are isolated from the blood remaining in theumbilical vein following child birth. This blood has been shown tocontain immature stem cells or progenitor cells.
Skeletal myoblasts are cells responsible for the regeneration ofskeletal muscle following injury. These cells have the ability tofuse with other myoblasts or damaged muscle fibers. In the presentinvention, skeletal myoblasts can be seeded into the scaffold sothat these cells can integrate into a host's damaged myocardium orother damaged muscle tissue, and improve tissue properties orfunctionally participate in contraction. MDCs are a population ofcells isolated from adult skeletal muscle that are similar tomyoblasts, and can also be used to seed scaffolds that are to beimplanted in areas of muscle damage. Go cells are also isolatedfrom adult skeletal muscle, and these non-satellite cells expressGATA-4 and, under in vitro growth conditions, develop intospontaneously beating cardiomyocyte-like cells.
When the scaffold is not seeded with cells prior to implantation ofthe scaffold, the scaffold serves as an in situ self-seedingimplant. After implantation of the scaffold into an area of tissuedamage, cells migrate into the scaffold and preferentially attachto the A-strips. The cells are aligned to the architecture of thescaffold. As the cells grow and differentiate, they are able torefashion the architecture of the scaffold such that the scaffoldis adopted into the surrounding tissue. When all the materials ofthe scaffold are biodegradable, the scaffold completely degradesleaving behind only the integrated cellular tissue andtissue-specific extracellular matrix.
The scaffold of the present invention can be designed to growand/or repair any cell or tissue type in vitro or in vivo. If, forexample, the scaffold is used to grow a skin substitute in vitro,the scaffold is designed so that the scaffold layers are randomlyaligned relative to each other. If, for example, the scaffold isused to repair and regenerate damaged myocardial tissue, then thescaffold can be designed such that the scaffold layers are allsubstantially aligned relative to each other in a parallelorientation.
Following implantation of the seeded or non-seeded scaffold into asubject, the subject can be treated with bioactive agents in orderto promote the proliferation and differentiation of cells in thescaffold. Preferred bioactive agents for subject treatment includeGM-CSF, G-CSF, IL-1, IL-3, SCF, VEGF, Flt-3 ligand, heme oxygenase,cell survival factors, and attachment factors such as collagen,laminin, fibronectin, and fragments and variants thereof whichretain the same biological activity including fusion proteins andchimeric proteins. Chemical agents, such as nitric oxide and5-azacytidine can also be used to promote engraftment of scaffolds.Alternatively, the scaffolds of the present invention can bepretreated to contain bioactive agents or chemical agents.
A subject to be treated according to the method of the invention isone who has suffered an injury or has an illness or disorder thatresults in damaged or diseased tissue. In certain cases, the injuryis an infarction that results in tissue necrosis, and moreparticularly, a myocardial infarction. Such subjects include humansand animals, such as laboratory animals or feed animals, including,but not limited to, mice, rats, rabbits, dogs, cats, cattle, swine,non-human primates, and others. Preferably, the subject is a human.
The scaffold can be used to treat any organ or tissue in needthereof. Examples of tissues include, but are not limited to, bone,cartilage, and striated muscle, which include cardiac muscle andskeletal muscle. Examples of organs that can be treated by themethods of the invention include, but are not limited to, heart,liver, brain, kidney, intestine, lung, eye, pancreas, bladder, andspinal cord.
The replacement of defective cardiac tissue by functioningmyocardium is desired. Two strategies for the repair of cardiactissue, the implantation of isolated cells and the implantation ofin vitro designed tissue equivalents, are both improved when usedwith the scaffolds of the present invention.
In prior studies, the implantation or injection of cells intomyocardial scar tissue improved global heart function (for example,see Carrier, R. L. et al., "Cardiac Tissue Engineering: CellSeeding, Cultivation Parameters, and Tissue ConstructCharacterization," Biotechnology and Bioengineering, 1999,64(5): 580-589; Folliguet, T. A., "Adult Cardiac MyocytesSurvive and Remain Excitable During Long-Term Culture on SyntheticSupports," J. Thorac. Cardiovasc. Surg., 2001, 121:510-519;Li, R. K. et al., "Cardiomyocytes transplantation improvesheart function," Annals of Thoracic Surgery, 1996, 62:654-661;Li, R. K. et al., "In vivo survival and function oftransplanted rat cardiomyocytes," Circulation Research, 1996,78:283-288; Li, R. K. et al., "Survival and Function ofBioengineered Cardiac Grafts," Circulation, 1999, 100 (suppl.II): 63-69; and Li, R. K. et al., "Construction of abioengineered cardiac graft," J. Thorac. Cardiovasc. Surg.,2000, 119:368-375. Surprisingly, the effect appeared to beindependent of cell origin, as positive results were reported fromfetal or neonatal cardiac myocytes, fibroblasts, endothelial cells,smooth muscle cells, skeletal myoblasts, and stem cells. Further,expanding autologous skeletal myoblasts ex vivo and injecting theminto the area of scar tissue has also led to some positive results.However, despite survival and differentiation of implanted cells,mechanical and electrical cell-cell contacts between graft andhost, a requirement for functioning myocardial tissue, is onlyrarely observed. Thus, one embodiment of the present inventioncomprises implanting a scaffold (either with or without pre-seedingof cells or bioactive agents) to the area of myocardial scartissue. Concurrently, subsequently or prior to implantation of thescaffold, cells are additionally implanted. The scaffolds of thepresent invention are designed such that mechanical and electricalcell-cell contacts between graft and host are improved.
The second approach for the repair/replacement of damaged ordiseased cardiac tissue uses in vitro designed cardiac constructs.However, prior in vitro designed cardiac constructs have sufferedthe problems of: (1) the scaffold materials exhibit an intrinsicstiffness that compromises diastolic function, (2) biodegradationof the scaffold materials remains incomplete, adding to problemswith diastolic function, (3) size limitation of engineeredconstructs due to a lack of metabolic or oxygen supply in the coreof three-dimensional constructs, and (4) lack of homogeneous celldistribution within the scaffold. As to the last problem, cardiacmyocytes seeded on or in gelatin meshes formed only a 300micrometer thick cell layer on the outside due to problems of themyocytes in migrating through the gelatin meshes (Li et al., 2000).Similarly, cardiac myocytes seeded on synthetic polymer scaffoldsonly form cell layers of 50 to 70 micrometers.
In one embodiment, the scaffolds of the present invention remedythe above-stated problems. Because the scaffold can have multiplescaffold layers with A-strips that promote cell-attachment, andbecause the separating layers and S-strips can be designed to havespecific pore sizes, homogeneous or even cell distribution withinthe scaffold is greatly improved over prior scaffolds.Additionally, the size is not as limited as prior constructs withthe present scaffolds because the pores within the scaffold allowfor the interchange of metabolites, oxygen, and bioactive agents.Further, in a preferred embodiment, the scaffolds of the inventionare made of completely biodegradable materials that allow thescaffold to be re-shaped and readily adopted into the surroundingtissue in which it is implanted.
C. Producing the Scaffold
The present invention also provides a method of producing asemi-solid, three-dimensional, biocompatible scaffold by layeringone or more sheets of biocompatible A-strip material by a fixeddistance from each other; cutting the sheets to provide A-strips ofa fixed width, maintaining the parallel A-strips at a fixeddistance from each other; and contacting the parallel A-strips witha biocompatible S-strip material under conditions to fill the spaceseparating the A-strips within a layer and between layers to form asemi-solid, three-dimensional, biocompatible and, preferably,biodegradable scaffold. This method of producing the scaffold ispreferred when the source material for producing A-strips isavailable in sheets, for example, submucosa. Biocompatiblematerials such as polyglycolic acid, polylactic acid, theircopolymers, poly(epsilon-caprolactone), polyhydroxybutyrate,polyester copolymers, polycarbonates, polyacrylates,polyanhydrides, polyorthoesters can be formed into sheets, forexample, by the methods reported in U.S. Pat. No. 5,723,508.A-strips can comprise synthetic materials which may be combinedwith a factor that encourages cell attachment.
Alternatively, when the source material for producing A-strips isnot available in sheets, a method of producing a semi-solid,three-dimensional, biocompatible scaffold is provided. For example,extracellular matrix gels and liquid matrices (including fluidizedsubmucosa) can be placed (for example, by micro-pipette) onto orinto gelatinous layers of alginate to form A-strips according tothe appropriate dimensions stated herein, thereby forming ascaffold layer. Also, proteins or peptides (for example, integrins,collagen, laminin, fibronectin, vitronectin, or RGD peptides) thatpromote cell attachment can be patterned as A-strips ontogelatinous layers of alginate, thereby forming a scaffold layer.These scaffold layers can be sandwiched between layers of alginate(i.e., separating layers) to form a three dimensional scaffold.This method provides an alternative strategy to producing scaffoldswith A-strips of cell-adhesive material obtained fromnon-reconstituted submucosa.
For scaffolds of the invention and the methods of producing thosescaffolds, the overall dimensions of the sheet are determined bythe intended uses of the scaffold. Scaffolds can be produced thatare larger for an area of intended implantation. For manufacturingpurposes, it is preferable to produce large scaffolds from whichsmaller scaffolds can be obtained.
When the cell-adhesive material is in the form of a sheet, thesheet can be coated with one or more of the bioactive agentspreviously described. When the cell-adhesive material is originallyin the form of a liquid, liquid matrix, or gel, one or morebioactive agents can be mixed with the cell-adhesive material priorto forming the strips. In addition, if desired, A-strips of anymaterial can be coated with a physiological solution comprising oneor more bioactive agents. Similarly, the S-strips and theseparating layers can be coated and/or mixed with one or morebioactive agents.
Claim 1 of 29 Claims
1. A semi-solid, three-dimensional biocompatible scaffold for cellgrowth, tissue repair or regeneration comprising more than onescaffold layer of alternating attachment-strips (A-strips) andseparating-strips (S-strips), wherein the A-strips within eachscaffold layer are aligned parallel to each other, and wherein theA-strips preferentially promote cellular attachment to A-stripsover cellular attachment to the S-strips.
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Abstract
The present invention relates to a biocompatible, three-dimensionalscaffold useful to grow cells and to regenerate or repair tissue inpredetermined orientations. The scaffold is particularly useful forregeneration and repair of cardiac tissue. The scaffold containslayers of alternating A-strips and S-strips, wherein the A-stripswithin each layer are aligned parallel to each other andpreferentially promote cellular attachment over attachment to theS-strips. Methods of producing and implanting the scaffold are alsoprovided.
Description of the Invention
SUMMARY OF THE INVENTION
The present invention is directed to a semi-solid,three-dimensional, biocompatible scaffold for cell growth, tissuerepair or regeneration, comprising one or more layers ofalternating attachment-strips ("A-strips") andseparating-strips ("S-strips") wherein the A-stripswithin each layer are aligned parallel to each other andpreferentially promote cellular attachment over attachment to theS-strips. The present invention is also directed to methods ofproducing and implanting such scaffolds.
In one aspect, the scaffold is biodegradable and re-shapeable.After implantation of the scaffold into an area of tissue damage ordisease, cells migrate into the scaffold and preferentially attachto the A-strips allowing cells to align in accordance with thearchitecture of the scaffold. The architecture of the scaffold canthus provide spatial- and orientation-dependent signals for cellgrowth and/or differentiation. As the cells grow and differentiate,they reshape the architecture of the scaffold such that thescaffold is adopted into the surrounding tissue. Eventually thesescaffolds completely degrade leaving behind the integrated cellulartissue and tissue-specific extracellular matrix.
The design of the scaffolds can be varied in relation to differenttissue architectures by varying the dimensions of the elements ofthe scaffolds, and/or the materials that the elements of thescaffold are made of. The elements of the scaffold are theA-strips, S-strips and the separating layers, and their dimensionsas to width, length, thickness and porosity are fixed according toa scaffold's intended use in the repair, regeneration or growth ofa particular tissue or cell type, in vivo or in vitro. The width ofthe A-strips and S-strips are fixed to promote the orderedalignment of cells within a layer so that cells can receivespatial- and orientation-dependent signals for cell growth and/ordifferentiation from each other and from the strip materials. Thematerials of the scaffold are also chosen such that the A-stripspromote the adhesion of cells more than the S-strips and theseparating layers, and such that the porosity of the S-strips andseparating layers allow a homogeneous distribution of cellsthroughout the scaffold. Further, the scaffold can be supplementedor treated with bioactive agents such as those, for example, thatpromote cell migration and proliferation.
In accordance with the invention, the scaffold can be implanted ina subject at a site of tissue damage without having been pre-seededwith cells. In this embodiment, the scaffold is seeded with cellsin situ either by endogenous cells or by cells separately implantedinto the subject. Alternatively, the scaffold can be pre-seededwith cells in vitro prior to implantation in a subject.
In another aspect of the present invention, methods of producingthe scaffolds of the inventions are provided. In one embodiment, amethod of producing a semi-solid, three-dimensional, biocompatiblescaffold comprises layering one or more sheets of biocompatiblestrip material by a fixed distance from each other; cutting thesheets to provide A-strips of a fixed width, maintaining theparallel A-strips at a fixed distance from each other; andcontacting the parallel A-strips with a biocompatible material thatdoes not promote cellular attachment under conditions to fill thespace separating the A-strips, thereby forming S-strips, and tofill the space separating the layers; to form a semi-solid,three-dimensional, biocompatible and, preferably, biodegradablescaffold.
DETAILED DESCRIPTION OF THE INVENTION
The issued U.S. patents, published and allowed applications, andreferences cited herein are hereby incorporated by reference.
The present invention relates to a biocompatible, three-dimensionalscaffold useful for growing cells, regenerating or repairingtissue. This invention also provides methods of producing andimplanting the scaffold. The semi-solid, three-dimensional,biocompatible scaffold for cell growth, tissue repair orregeneration, or any combination thereof, comprises one or morelayers of alternating attachment-strips ("A-strips") andseparating-strips ("S-strips"), wherein the A-stripswithin each layer are aligned parallel to each other, and whereinthe A-strips preferentially promote cellular attachment."Attachment-strips" or "A-strips" are definedherein as an element of the scaffold that preferentially promotescellular attachment over other elements of the scaffold."Separating-strips" or "S-strips" are definedherein as an element of the scaffold that does not preferentiallypromote cellular attachment, and in relation to the architecture ofthe scaffold, serves to separate the A-strips from each otherwithin a scaffold layer.
The dimensions and the materials of the scaffold are designed toallow cells to align in a manner to receive spatial- and/ororientation-dependent cues or signals from other cells and/or fromthe materials that constitute the A-strips of the scaffold. Thesesignals or cues help the cells in the scaffold to grow anddifferentiate so that the cells can adopt to and/or integrate intothe surrounding tissue in which the scaffold is implanted.Additionally, the scaffold can also provide the cues or signalssuch that cells in the scaffold grow and differentiate intofunctional tissues in vitro.
A. Structure and Materials of the Scaffold
The scaffold of the present invention has from one to many layers.Preferably the scaffold comprises multiple alternating layers ofscaffold layers and separating layers. The scaffold layer iscomposed of alternating A-strips and S-strips where the A-stripswithin the scaffold layer are aligned parallel to each other. Inaccordance with the invention, A-strips preferentially promote theattachment of cells, and S-strips and separating layers do notpreferentially promote the attachment of cells. As used herein,"preferentially promotes" means that more cells attach tothe A-strips rather than to the S-strips or the separating layers.In addition, preferentially promoting attachment can be achieved bypreferential proliferation or differentiation of the cells on theA-strips rather than on the S-strips or the separating layers.
It is preferred that greater than about 70% of the cells within ascaffold are present on A-strips. It is more preferred that greaterthan about 90% of the cells within a scaffold are present onA-strips. The percentage of cells present on A-strips as comparedto S-strips can be tested by performing a 2-dimensional cellculture experiment where the culture surface is coated with S-stripand A-strip material in an area and design similar to one layer ofa desired scaffold. A cell type will be used which is relevant tothe intended target tissue of the scaffold.
The A-strips can range from about 20 micrometers to about 200micrometers in thickness, and from about 20 micrometers to about100 micrometers in width. These dimensions, especially the width,are relevant to the cell-attachment properties of the A-strip. Forexample, if the A-strips are too narrow (i.e., if the A-strips aremore narrow than the size of a cell), then cells cannot properlyattach. If the A-strips are too wide, then cells may not properlyor efficiently align in relation to the architecture of the layerbecause the cells cannot align on the A-strips in a linear fashion.For use with cardiomyocytes, the preferred width of the A-strips isfrom about 20 to about 50 micrometers in width.
The S-strips can range from about 20 micrometers to about 200micrometers in thickness, and from about 20 micrometers to about200 micrometers in width. If the S-strips are too narrow, i.e.,similar to the width of a single cell, then the A-strips cannotfunction to align the cells in a parallel orientation. For example,if the cells on one A-strip are able to span the distance of theS-strip to another A-strip, then the A-strips are less effective inaligning cells in a substantially linear fashion on an A-strip. Forcardiomyocytes, the preferred width of an S-strip is from about 50to about 100 micrometers in width.
The separating layer alternates with the scaffold layer. As definedherein, a "scaffold layer" is a layer of a scaffoldcomprising alternating A-strips and S-strips. In other words, theseparating layer serves to separate the scaffold layers from eachother. According to the present invention, a separating layer canbe made of a material suitable for a S-strip. Hence, a separatinglayer is made of a material that does not preferentially promotecell attachment and/or proliferation and/or differentiation. Thus,the A-strips of the scaffold preferentially promote cell attachmentover the S-strips and the separating layers.
The separating layer can range from about 20 micrometers to about200 micrometers in thickness. Further, the separating layer and theS-strips can, but need not necessarily contain pores. The poresallow cell migration and can range from about 10 micrometers toabout 300 micrometers in diameter. The pore size cannot exceed thedimensions of a layer. The pores allow cells to migrate throughoutthe scaffold, to aid in providing an even or homogeneousdistribution of cells throughout the scaffold. The pores alsoenable the migration of soluble peptides and proteins that arenecessary for cellular growth and communication. The pores alsoenable efficient vascularization between the scaffold and thesurrounding tissue.
The alignment of the A-strips within a scaffold layer is parallel.However, when the scaffold is multi-layered, the alignment of theA-strips between successive scaffold layers need not be parallel.For example, the scaffold can be constructed such that there is nooverall alignment of the A-strips from scaffold layer to scaffoldlayer. This overall random alignment serves to grow and regeneratetissue where cell alignment is not ordered, for example, in skin.Of course, the scaffold can be constructed such that all theA-strips are aligned in one general parallel orientation, or in anyother desired pattern, e.g., such as alternating 90.degree. angles.
The scaffold is preferably composed of a material that permitscells to reshape the scaffold. Such properties allow the cells ofthe scaffold to become integrated with or adopted into thesurrounding tissue in which the scaffold is implanted. By"integrating" or "adopting", the cells of thescaffold are not rejected by the surrounding tissue in which thescaffold is implanted. The scaffold is more re-shapeable when thematerials of the scaffold are biodegradable and not synthetic.
The A-strips of the present invention are constructed from abiocompatible substance that can be formed into a shape thatessentially resembles a strip or a fiber having the dimensionsstated herein. Biocompatible substances that can be used to formthe A-strips, include but are not limited to, extracellular matrixmaterial, proteins or peptides that are not present inextracellular matrices but have cell attachment properties, andsynthetic materials, provided such materials can be formed intoA-strips of the appropriate size and have the requisite biologicalcharacteristics to preferentially promote cell attachment and/orproliferation and/or differentiation. Non-synthetic, biocompatibleand biodegradable materials are preferred for A-strips, such asextracellular matrix material. If synthetic materials are used asstrip material, the synthetic materials can be combined with eitherextracellular matrix material or proteins or peptides withcell-attachment properties so that the strips preferentiallypromote cell attachment or proliferation.
According to the present invention, "extracellular matrixmaterial" is any material or substance that is present in anextracellular matrix. An extracellular matrix is an acellular sheetor layer that consists of three major classes of biomolecules: (1)structural proteins such as collagen and elastin, (2) specializedproteins such as, perlacan, agrin, laminin, fibronectin, entactin,nidogen, and fibrillin, and (3) proteoglycans which are composed ofa protein core to which is attached long chains of repeatingdisaccharide units (glycosaminoglycans (GAGs)). Thus, according tothe present invention, any of the above-types of biomolecules,individually or in combination, in natural, processed orrecombinant forms, are considered extracellular matrix material.
The extracellular matrix material can be isolated from animals orfrom an in vitro cellular source rich in producing extracellularmatrix material. Sheets of extracellular matrix isolated fromanimals can be cut to form the A-strips of the scaffold.Alternatively, sheets of isolated extracellular matrix material canbe processed into gels or liquids (see for example, U.S. Pat. No.5,275,826 or U.S. Pat. No. 4,829,000), and these gels or liquidscan be molded to form the A-strips of the scaffold. Additionally,recombinant forms of extracellular matrix materials can be used toform the strips of the scaffold.
The extracellular matrix material can be isolated from a variety oftissue sources, for example, essentially any submucosa. A submucosais a layer of areolar connective tissue that lies beneath themucosa. Examples of submucosa that can be used as a source ofextracellular matrix material, include intestine submucosa, stomachsubmucosa, liver submucosa, and urinary bladder submucosa. Basementmembranes can also be used as a source of extracellular matrixmaterial. Basement membranes are thin, but continuous sheets thatseparate epithelium from stroma and surround nerves, muscle fibers,smooth muscle cells and fat cells. Additionally, tissues such asbone, bone marrow, cartilage and placenta can also be sources ofextracellular matrix material. Further, embryonic and fetal cardiactissue can be sources of extracellular matrix material.Representative processes that can be used to isolate extracellularmatrix material from tissue are described in, Voytik-Harbin, S. L.,"Three-dimensional extracellular matrix substrates for cellculture," Methods Cell Biol., (2001), 63:561-81.
The tissue sources of extracellular matrix material can be isolatedfrom any mammal, including but not limited to: pig, human, cow,sheep, goat, donkey, horse, rabbit, dog, cat, rat and mouse. Theextracellular matrix material can be in the form of a sheet fromwhich A-strips are cut, or the material can be processed into theirseparate components or combinations of components, and then formedinto A-strips or a sheet. Such sheets can then be cut into A-stripsin accordance with the invention. Alternatively, if individualA-strips are formed, these A-strips have the dimensions as statedherein and are arrangeable in a parallel orientation.
Sheets of extracellular matrix can be isolated from essentially anytype of submucosa. For example, in U.S. Pat. No. 4,902,508, bydelaminating the tunica muscularis and at least the luminal portionof the tunica mucosa of the small intestine, a tunica submucosa wasisolated. As described in U.S. Pat. No. 6,099,567, the stomachsubmucosa can be isolated by delaminating the smooth muscle layersof the muscularis externa and at least the luminal portion of themucosal layer of a segment of the stomach. As described in U.S.Pat. No. 5,554,389, urinary bladder submucosa is isolated bydelaminating the abluminal muscle cell layers and at least theluminal portion of the mucosal layer of a segment of urinarybladder. Acellular and detoxified sheets of submucosa arecommercially available, for example, see COOK.RTM. BiotechIncorporated, SIS.TM. Technology (COOK Biotech Incorporated, 3055Kent Avenue, West Lafayette, Ind. 47906 USA).
Extracellular matrix material can also be isolated as a slurry orsolution. Solutions or slurries can be processed or molded intoA-strips of the appropriate dimensions, for example, by thefollowing processes: freeze-drying in a mold; air drying intoA-strips; air drying into sheets which are cut into A-strips;gelled by neutralizing pH; and solutions or slurries can becombined with a second material (e.g., collagen, gelatin,self-assembling peptides) which can be solidified, cross-linked, orotherwise turned into a solid without affecting the biologicalactivity of the extracellular matrix material.
Processed or recombinant forms of extracellular matrix materialscan be used as A-strip material. For example, U.S. Pat. No.4,829,000 reports a processed, reconstituted,basement-membrane-derived extracellular matrix composition(MATRIGEL.TM.). Also, U.S. Pat. No. 5,275,826 reports fluidizedforms of submucosa. Purified or recombinant forms of collagen,elastin, perlacan, agrin, laminin, fibronectin, entactin, nidogen,fibrillin, proteoglycans can be used as strip material, and arecommercially available. For example, BD BIOSCIENCES.TM. offers avariety of ECM products that can be used in the present invention:BD Matrigel.TM. Basement Membrane Matrix, Collagen I ECM, CollagenIII ECM, Collagen IV ECM, Collagen V ECM, Fibronectin ECM, LamininECM.
The extracellular matrix material can be from a syngeneic,allogeneic or xenogeneic source. When used as the materialcomprising the A-strips of the scaffold, the extracellular matrixmaterial is acellular, and should not be recognized as"foreign" by a host's immune system, and therefore shouldnot be rejected by the host. Further, the biocompatible materialsthat comprise the scaffold are substantially endotoxin free.
Proteins and peptides that have cell-attachment properties but arenot present in extracellular matrices can also be used as A-stripmaterial. Such proteins and peptides include, but are not limitedto, vitronectin, polypeptides with the amino acid sequencearginine-glycine-aspartic acid (`RGD sequence`), and poly-L-lysine.According to the present invention, proteins and peptides that havecell attachment properties are proteins or peptides that bind tomolecules on a cell surface with an affinity such that the bindingis not transient.
Synthetic materials such as polylactic acid (PLA), poly-glycolicacid (PGA), co-poly-lactic/poly-glycolic acid polymers (PLGA), canalso be used as A-strip material, if a non-biodegradable scaffoldis desired. If synthetic materials are used as A-strip material,the synthetic materials can be combined with either extracellularmatrix material or proteins or peptides with cell-attachmentproperties so that the A-strips preferentially promote cellattachment. Preferably, the A-strips of the present invention aremade from extracellular matrix material or proteins or peptideswith cell attachment properties.
The S-strips and the separating layers of the invention arebiocompatible and preferably biodegradable substances that do notpreferentially promote cell attachment. Preferably, the S-stripsand the separating layers do not permit cells to substantiallyattach. The material of the S-strips and the material of theseparating layer do not have to be identical. Examples of S-stripsand separating layer material include, but are not limited to,alginate, agarose, polylactic acid (PLA), poly-glycolic acid (PGA),co-poly-lactic/poly-glycolic acid polymers (PLGA), gelatin,ethylene-vinyl acetate, fibrin, sucrose octasulfate, dextran,polyethylene glycol, polyacrlyamide, cellulose, latex,polyhydroxyethylmethacrylate, nylon, Dacron,polytetrafluoro-ethylene, polystyrene, polyvinylchlorideco-polymer, cat gut, cotton, linen, polyester, and silk. Alginate,agarose and gelatin, are preferred. It is also preferred that allof the elements of the scaffold have a similar rate ofbiodegradation.
When the S-strips and separating layers comprise alginate, theporosity of alginate can be manipulated by using different sourcesof alginate, by varying the concentration of alginate, and byvarying the divalent cations used to help polymerize the alginate.
Proteins or peptides can be micro-patterned on the surface ofbiocompatible sheets, A-strips, S-strips or separating layers. Inthe present invention, proteins or peptides are `micro-patterned`by immobilizing proteins or peptides in micrometer sized shapes onthe surface of biocompatible materials.
Advances in patterning technology have generated a range oftechniques with which biomolecules can be immobilized on surfaceswith microscale precision. Some techniques that can be used withthe present invention include microcontact printing,photolithography, photochemistry, 3D printing, and microwriting.These techniques are known to those of skill in the art.
The scaffold of the present invention can also be treated withbioactive agents. These agents can be used alone or in combinationand include, but are not limited to, vascularization-promotingfactor, a cytokine, a growth factor, an enzyme, a hormone, anangiogenesis factor, a vaccine antigen, an antibody, a clottingfactor, a regulatory protein, a transcription factor, a receptor, astructural protein, and any functional fragment, variant orcombinations thereof. Preferably, the agents are located on or inthe A-strips of the scaffold, although they can be present in theS-strips and the separating layers, provided they do not alter theoverall characteristic of the scaffold to promote cellularattachment to the A-strips. Agents that recruit cells or directcells include cytokines, growth factors, enzymes, hormones,angiogenesis factors, regulatory proteins, transcription factors,receptors, structural proteins and any of their bioactivefunctional fragments. Agents that promote cell attachment includereceptors and structural proteins. Agents that promotevascularization of the scaffold and tissue include vascularizationpromoting factors, cytokines, growth factors and angiogenesisfactors.
Examples of specific bioactive agents include, but are not limitedto, collagen, laminin, fibronectin, granulocyte-colony stimulatingfactors (G-CSF), granulocyte-macrophage colony stimulating factor(GM-CSF), stem cell factor (SCF), vascular endothelial growthfactor (VEGF), platelet-derived growth factor (PDGF), transforminggrowth factors, human growth hormone (hGH), Factor VIII, Factor IX,erthropoietin (EPO), albumin, heme oxygenase, hemoglobin, alpha-1antitrypsin, calcitonin, glucocerebrosidase, low densitylipoprotein (LDL) receptor, IL-2 receptor, globins,immunoglobulins, catalytic antibodies, interleukins, chemokines,insulin, insulin-like growth factor 1 (IGF-1), insulinotropin,parathyroid hormone (PTH), leptin, an interferon, nerve growthfactors, epidermal growth factor (EGF), endothelial cell growthfactor, endothelial cell stimulating angiogenesis factor (ESAF),angiogenin, tissue plasminogen activator (t-PA), folliclestimulating hormone (FSH), Flt-3 ligand, megakaryocyte growth anddevelopment factor (MGDF), and 3-hydroxy-3-methyl glutaryl coenzymeA (HMG CoA) reductase inhibitors, and any functional fragment,variant or combinations thereof.
The architecture of the scaffold helps in promoting cellular growthand differentiation. Most cells can grow in vitro without anyscaffolding. Yet, many cells do not fully differentiate withoutspatially defined cell-contact and orientation signals. Forexample, integrin receptors on the surface of cells bind to RGD(arginine-glycine-aspartic acid) sequences in extracellular matrixproteins such as fibronectin. This interaction induces cellspreading and intracellular signaling. Spatial orientation helps toprovide proper cell-to-cell signaling and cellular function. Forexample, cardiomyocytes are organized into parallel cardiac musclefibers with intracellular contractile myofibrils oriented parallelto the long axis of each cell and junctional complexes betweenabutting cells concentrated at the ends of each cardiomyocyte.Without this highly oriented architecture, electromechanicalcoupling of cardiomyoctes does not occur, and the transmission ofdirected contraction over long distances is not possible. Thus,although cardiomyocytes can be grown in in vitro cultures, thecultured cardiomyocytes only spread to form an epithelioid sheet,with disorganized myofibrils and diffuse intercellular junctions.
B. Cellular Growth, Tissue Repair and Regeneration
The scaffolds of the present invention are used for both in vitroand in vivo cellular growth and differentiation. The purpose of invivo use is for the repair and regeneration of damaged and/ordiseased tissue. The scaffolds can be used as self-seedingscaffolds for cellular attachment, growth and repair in situ; orthe scaffold can be seeded in vitro with cells prior toimplantation. Generally, cells at a concentration of1.times.10.sup.6 to 5.times.10.sup.6 cells/ml are added to a dishor bioreactor containing a scaffold(s), and the cells are incubatedfor 24-72 hours at 37.degree. C. at 5% CO.sub.2 for initialseeding. However, the cell concentration can range from about1.times.10.sup.4 to about 1.times.10.sup.7, or higher for cellsthat can achieve such concentrations.
As used herein, the term "damaged or diseased tissue"means tissue in which cells have been lost or have died due toinsufficient blood supply, mechanical injury, infection,irradiation, trauma, disease, or other insult. For example, damagedand or diseased tissue includes, but is not limited to, scartissue, and tissue that is torn, crushed, or has undergone necrosisresulting from blood loss. By "necrosis" is meantpathologic cell death following irreversible damage to the cell.The damaged or diseased tissue can be distinguished from thesurrounding tissue, for example, by physical inconsistency ordiscontinuity.
Cells which can used to seed the scaffold in vitro include, but arenot limited to, side population (SP) adult stem cells, Lineagenegative (Lin.sup.-) stem cells, Lin.sup.-CD34.sup.- stem cells,Lin.sup.-CD34.sup.+ stem cells, Lin.sup.-cKit.sup.+ stem cells,mesenchymal stem cells (MSC), cord blood cells, tissue stem cells,whole bone marrow, bone marrow mononuclear cells (BM-MNCs), cardiacstem cells, tissue stem cells, endothelial progenitor cells (EPCs),skeletal myoblasts (satellite cells), muscle derived cells (MDCs),go cells, endothelial cells, adult cardiomyocytes, fibroblasts,smooth muscle cells, genetically modified cells, MyoD scarfibroblasts, pacing cells, embryonic stem cell clones, embryonicstem cells, fetal or neonatal cells, teratoma cells, and anycombination thereof.
SP cells can be isolated by a fluorescence-activated cell sorter(FACS) technique utilizing the ability of SP cells to excludeHoechst dye from the nucleus. In addition to bone marrow, SP cellshave been isolated from most tissues, including cardiac andskeletal muscle. By the more common surface identification, thesecells are Lin.sup.-, Sca-1.sup.+, c-Kit.sup.+, CD43.sup.+'CD45.sup.+' CD34.sup.-.
Bone marrow cells are isolated, and all the cells that havedifferentiated to a specific lineage are removed, either by FACSsorting or magnetic-bead sorting. The cells that remain are thestem and progenitor cells, or Lin.sup.- cells. Of these cells, themost primitive bone marrow-derived stem cells areLin.sup.-CD34.sup.-. Cells that are Lin.sup.-CD34.sup.+ includehematopoietic stem cells. Lin.sup.-cKit.sup.+ cells express cKit,which is the cell surface receptor for stem cell factor. Therefore,Lin.sup.-cKit.sup.+ cells are often used as a stem cell population.Lin.sup.-cKit.sup.+ cells have been isolated from the heart and thebone marrow.
Whole bone marrow can be used for transplantation and scaffoldseeding. The whole bone marrow sample is filtered to remove boneparticles. Whole bone marrow includes many extracellular matrixproteins and growth factors. The BM-MNC population can be separatedfrom whole bone marrow by a density gradient centrifugationprocedure. The BM-MNC population contains non-granular white bloodcells, progenitor cells, and stem cells. Although most work to datehas focused on isolating stem cells from the bone marrow, some stemcells (e.g., SP, Lin.sup.-cKit.sup.+) can be isolated from tissues,for example fat and cardiac muscle.
MSCs are stem cells that ordinarily differentiate into the cells ofmesenchymal tissues (e.g. bone, cartilage, fat), but can alsodifferentiate into cardiomyocytes under certain conditions. MSCsare isolated from the bone marrow, and have the capacity toproliferate in vitro. EPCs are also isolated from the bone marrowbased on cell surface markers. EPCs differentiate into endothelialcells, and when transplanted to ischemic tissue, these cells canform new blood vessels.
Cord blood cells are isolated from the blood remaining in theumbilical vein following child birth. This blood has been shown tocontain immature stem cells or progenitor cells.
Skeletal myoblasts are cells responsible for the regeneration ofskeletal muscle following injury. These cells have the ability tofuse with other myoblasts or damaged muscle fibers. In the presentinvention, skeletal myoblasts can be seeded into the scaffold sothat these cells can integrate into a host's damaged myocardium orother damaged muscle tissue, and improve tissue properties orfunctionally participate in contraction. MDCs are a population ofcells isolated from adult skeletal muscle that are similar tomyoblasts, and can also be used to seed scaffolds that are to beimplanted in areas of muscle damage. Go cells are also isolatedfrom adult skeletal muscle, and these non-satellite cells expressGATA-4 and, under in vitro growth conditions, develop intospontaneously beating cardiomyocyte-like cells.
When the scaffold is not seeded with cells prior to implantation ofthe scaffold, the scaffold serves as an in situ self-seedingimplant. After implantation of the scaffold into an area of tissuedamage, cells migrate into the scaffold and preferentially attachto the A-strips. The cells are aligned to the architecture of thescaffold. As the cells grow and differentiate, they are able torefashion the architecture of the scaffold such that the scaffoldis adopted into the surrounding tissue. When all the materials ofthe scaffold are biodegradable, the scaffold completely degradesleaving behind only the integrated cellular tissue andtissue-specific extracellular matrix.
The scaffold of the present invention can be designed to growand/or repair any cell or tissue type in vitro or in vivo. If, forexample, the scaffold is used to grow a skin substitute in vitro,the scaffold is designed so that the scaffold layers are randomlyaligned relative to each other. If, for example, the scaffold isused to repair and regenerate damaged myocardial tissue, then thescaffold can be designed such that the scaffold layers are allsubstantially aligned relative to each other in a parallelorientation.
Following implantation of the seeded or non-seeded scaffold into asubject, the subject can be treated with bioactive agents in orderto promote the proliferation and differentiation of cells in thescaffold. Preferred bioactive agents for subject treatment includeGM-CSF, G-CSF, IL-1, IL-3, SCF, VEGF, Flt-3 ligand, heme oxygenase,cell survival factors, and attachment factors such as collagen,laminin, fibronectin, and fragments and variants thereof whichretain the same biological activity including fusion proteins andchimeric proteins. Chemical agents, such as nitric oxide and5-azacytidine can also be used to promote engraftment of scaffolds.Alternatively, the scaffolds of the present invention can bepretreated to contain bioactive agents or chemical agents.
A subject to be treated according to the method of the invention isone who has suffered an injury or has an illness or disorder thatresults in damaged or diseased tissue. In certain cases, the injuryis an infarction that results in tissue necrosis, and moreparticularly, a myocardial infarction. Such subjects include humansand animals, such as laboratory animals or feed animals, including,but not limited to, mice, rats, rabbits, dogs, cats, cattle, swine,non-human primates, and others. Preferably, the subject is a human.
The scaffold can be used to treat any organ or tissue in needthereof. Examples of tissues include, but are not limited to, bone,cartilage, and striated muscle, which include cardiac muscle andskeletal muscle. Examples of organs that can be treated by themethods of the invention include, but are not limited to, heart,liver, brain, kidney, intestine, lung, eye, pancreas, bladder, andspinal cord.
The replacement of defective cardiac tissue by functioningmyocardium is desired. Two strategies for the repair of cardiactissue, the implantation of isolated cells and the implantation ofin vitro designed tissue equivalents, are both improved when usedwith the scaffolds of the present invention.
In prior studies, the implantation or injection of cells intomyocardial scar tissue improved global heart function (for example,see Carrier, R. L. et al., "Cardiac Tissue Engineering: CellSeeding, Cultivation Parameters, and Tissue ConstructCharacterization," Biotechnology and Bioengineering, 1999,64(5): 580-589; Folliguet, T. A., "Adult Cardiac MyocytesSurvive and Remain Excitable During Long-Term Culture on SyntheticSupports," J. Thorac. Cardiovasc. Surg., 2001, 121:510-519;Li, R. K. et al., "Cardiomyocytes transplantation improvesheart function," Annals of Thoracic Surgery, 1996, 62:654-661;Li, R. K. et al., "In vivo survival and function oftransplanted rat cardiomyocytes," Circulation Research, 1996,78:283-288; Li, R. K. et al., "Survival and Function ofBioengineered Cardiac Grafts," Circulation, 1999, 100 (suppl.II): 63-69; and Li, R. K. et al., "Construction of abioengineered cardiac graft," J. Thorac. Cardiovasc. Surg.,2000, 119:368-375. Surprisingly, the effect appeared to beindependent of cell origin, as positive results were reported fromfetal or neonatal cardiac myocytes, fibroblasts, endothelial cells,smooth muscle cells, skeletal myoblasts, and stem cells. Further,expanding autologous skeletal myoblasts ex vivo and injecting theminto the area of scar tissue has also led to some positive results.However, despite survival and differentiation of implanted cells,mechanical and electrical cell-cell contacts between graft andhost, a requirement for functioning myocardial tissue, is onlyrarely observed. Thus, one embodiment of the present inventioncomprises implanting a scaffold (either with or without pre-seedingof cells or bioactive agents) to the area of myocardial scartissue. Concurrently, subsequently or prior to implantation of thescaffold, cells are additionally implanted. The scaffolds of thepresent invention are designed such that mechanical and electricalcell-cell contacts between graft and host are improved.
The second approach for the repair/replacement of damaged ordiseased cardiac tissue uses in vitro designed cardiac constructs.However, prior in vitro designed cardiac constructs have sufferedthe problems of: (1) the scaffold materials exhibit an intrinsicstiffness that compromises diastolic function, (2) biodegradationof the scaffold materials remains incomplete, adding to problemswith diastolic function, (3) size limitation of engineeredconstructs due to a lack of metabolic or oxygen supply in the coreof three-dimensional constructs, and (4) lack of homogeneous celldistribution within the scaffold. As to the last problem, cardiacmyocytes seeded on or in gelatin meshes formed only a 300micrometer thick cell layer on the outside due to problems of themyocytes in migrating through the gelatin meshes (Li et al., 2000).Similarly, cardiac myocytes seeded on synthetic polymer scaffoldsonly form cell layers of 50 to 70 micrometers.
In one embodiment, the scaffolds of the present invention remedythe above-stated problems. Because the scaffold can have multiplescaffold layers with A-strips that promote cell-attachment, andbecause the separating layers and S-strips can be designed to havespecific pore sizes, homogeneous or even cell distribution withinthe scaffold is greatly improved over prior scaffolds.Additionally, the size is not as limited as prior constructs withthe present scaffolds because the pores within the scaffold allowfor the interchange of metabolites, oxygen, and bioactive agents.Further, in a preferred embodiment, the scaffolds of the inventionare made of completely biodegradable materials that allow thescaffold to be re-shaped and readily adopted into the surroundingtissue in which it is implanted.
C. Producing the Scaffold
The present invention also provides a method of producing asemi-solid, three-dimensional, biocompatible scaffold by layeringone or more sheets of biocompatible A-strip material by a fixeddistance from each other; cutting the sheets to provide A-strips ofa fixed width, maintaining the parallel A-strips at a fixeddistance from each other; and contacting the parallel A-strips witha biocompatible S-strip material under conditions to fill the spaceseparating the A-strips within a layer and between layers to form asemi-solid, three-dimensional, biocompatible and, preferably,biodegradable scaffold. This method of producing the scaffold ispreferred when the source material for producing A-strips isavailable in sheets, for example, submucosa. Biocompatiblematerials such as polyglycolic acid, polylactic acid, theircopolymers, poly(epsilon-caprolactone), polyhydroxybutyrate,polyester copolymers, polycarbonates, polyacrylates,polyanhydrides, polyorthoesters can be formed into sheets, forexample, by the methods reported in U.S. Pat. No. 5,723,508.A-strips can comprise synthetic materials which may be combinedwith a factor that encourages cell attachment.
Alternatively, when the source material for producing A-strips isnot available in sheets, a method of producing a semi-solid,three-dimensional, biocompatible scaffold is provided. For example,extracellular matrix gels and liquid matrices (including fluidizedsubmucosa) can be placed (for example, by micro-pipette) onto orinto gelatinous layers of alginate to form A-strips according tothe appropriate dimensions stated herein, thereby forming ascaffold layer. Also, proteins or peptides (for example, integrins,collagen, laminin, fibronectin, vitronectin, or RGD peptides) thatpromote cell attachment can be patterned as A-strips ontogelatinous layers of alginate, thereby forming a scaffold layer.These scaffold layers can be sandwiched between layers of alginate(i.e., separating layers) to form a three dimensional scaffold.This method provides an alternative strategy to producing scaffoldswith A-strips of cell-adhesive material obtained fromnon-reconstituted submucosa.
For scaffolds of the invention and the methods of producing thosescaffolds, the overall dimensions of the sheet are determined bythe intended uses of the scaffold. Scaffolds can be produced thatare larger for an area of intended implantation. For manufacturingpurposes, it is preferable to produce large scaffolds from whichsmaller scaffolds can be obtained.
When the cell-adhesive material is in the form of a sheet, thesheet can be coated with one or more of the bioactive agentspreviously described. When the cell-adhesive material is originallyin the form of a liquid, liquid matrix, or gel, one or morebioactive agents can be mixed with the cell-adhesive material priorto forming the strips. In addition, if desired, A-strips of anymaterial can be coated with a physiological solution comprising oneor more bioactive agents. Similarly, the S-strips and theseparating layers can be coated and/or mixed with one or morebioactive agents.
Claim 1 of 29 Claims
1. A semi-solid, three-dimensional biocompatible scaffold for cellgrowth, tissue repair or regeneration comprising more than onescaffold layer of alternating attachment-strips (A-strips) andseparating-strips (S-strips), wherein the A-strips within eachscaffold layer are aligned parallel to each other, and wherein theA-strips preferentially promote cellular attachment to A-stripsover cellular attachment to the S-strips.
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